Biological Amyloids Chemically Damage DNA
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Biological Amyloids Chemically Damage DNA
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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2025, 16, 3, 355–364
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https://doi.org/10.1021/acschemneuro.4c00461
Published January 9, 2025

Copyright © 2025 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Amyloid fibrils are protein polymers noncovalently assembled through β-strands arranged in a cross-β structure. Biological amyloids were considered chemically inert until we and others recently demonstrated their ability to catalyze chemical reactions in vitro. To further explore the functional repertoire of amyloids, we here probe if fibrils of α-synuclein (αS) display chemical reactivity toward DNA. We demonstrate that αS amyloids bind DNA at micromolar concentrations in vitro. Using the activity of DNA repair enzymes as proxy for damage, we unravel that DNA-amyloid interactions promote chemical modifications, such as single-strand nicks, to the DNA. Double-strand breaks are also evident based on nanochannel analysis of individual long DNA molecules. The amyloid fold is essential for the activity as no DNA chemical modification is detected with αS monomers. In a yeast cell model, there is increased DNA damage when αS is overexpressed. Chemical perturbation of DNA adds another chemical reaction to the set of activities emerging for biological amyloids. Since αS amyloids are also found in the nuclei of neuronal cells of Parkinson’s disease (PD) patients, and increased DNA damage is a hallmark of PD, we propose that αS amyloids contribute to PD by direct chemical perturbation of DNA.

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Copyright © 2025 The Authors. Published by American Chemical Society

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1. Introduction

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Amyloids are long, ordered polymers of monomeric protein units noncovalently assembled through β-strands arranged perpendicularly to the fibril long axis forming a cross-β structure. (1) The cross-β arrangement is the basis of all amyloid fibers, but the exact packing (fold, topology) of the β-strand arrangement in each perpendicular plane varies widely among amyloid systems; even the same polypeptide can adopt different amyloid polymorphs depending on conditions and other unknown factors. (2) Many (maybe all) proteins can assemble into amyloids at extreme solvent conditions in vitro (1) and, therefore, amyloid formation is viewed as an intrinsic property of polypeptide chains. Although several functional amyloids are known (e.g., bacterial curli), (3,4) amyloid formation is mostly connected to human neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease, and type-2 diabetes. (5−8) Here, proteins with normal functions as monomers start (for some unknown reason) to assemble into amyloids, resulting in both loss of monomer function as well as gain of toxicity coupled to the assembly processes. Today, we know of over 50 diseases linked to aberrant amyloid formation. (1)
Here we focus on the amyloidogenic protein in PD but, due to the general nature of amyloids, our observations may be extended to other amyloid systems. In PD patients, amyloid fibers of the synaptic, 140-residue protein α-synuclein (αS) accumulate in cytosolic inclusions, called Lewy bodies, in dopamine neurons along with death of such neurons in the substantia nigra. (9) The cytoplasmic Lewy pathology is accompanied by genome instability in PD patients, animal models and cell cultures. (10−12) Notably, most diseases involving amyloids include also genome instability as another hallmark. Although accumulation of DNA damage is recognized as a primary hallmark of general aging, (13) PD patients and corresponding model systems display increased single-strand and double-strand DNA breaks. (11,14−16) In fact, studies have demonstrated that increased DNA damage may be one of the earliest events detectable in neurodegenerative diseases such as PD. (15)
In addition to the cytoplasm, there is a significant fraction of αS in the cell nucleus, and functional roles in DNA repair, nucleocytoplasmic transport, and regulation of gene transcription have been proposed. (17−19) However, most reports on nuclear αS suggest activities related to dysfunction. (12,20) The amount of αS in the nucleus appears to be increased by αS post-translational modifications, αS pathological mutations, and chemical insults to the cells. (21−24) Increased levels of αS in the nucleus were reported to perturb transcription of a master transcription activator (25) and chromatin-bound αS was correlated with DNA breaks. (26) Several studies have shown αS monomers to interact with DNA in vitro. (27,28) We recently demonstrated that monomeric αS binding increases the persistence length of DNA, (29) whereas binding of a truncated (pathological) variant of αS promotes DNA compaction. (30) In addition to nuclear αS monomers, nuclear Lewy pathology, i.e., αS amyloids in the nucleus, has repeatedly been noted in PD patients as well as animal models. (31) However, the consequences of αS amyloids in the nucleus remain unclear. (32−34)
Amyloid toxicity is often attributed to the ability to seed new amyloids, to translocate between cells, and to sterically block cellular functions. Amyloids have always been considered chemically inert until this was challenged when we showed that αS amyloids catalyze hydrolysis of ester and phosphoester bonds in vitro. (35,36) In addition, we detected distinct chemical alterations of important metabolites in neuronal cell lysates (devoid of proteins; only small molecules present) upon incubation with αS amyloids. (37) This enzyme-like behavior of αS amyloids, which has been paralleled by similar results on amyloid-β (linked to Alzheimer’s disease) and glucagon (hormone, unknown link to disease) amyloids, (38,39) implies that many amyloid systems may have yet-unknown chemical reactivities. (35)
Here we test the hypothesis that αS amyloid interactions with DNA are directly responsible (at least in part) for the widespread DNA damage observed in PD patients. By combining in vitro bulk and single-molecule biophysical and biochemical experiments, we reveal that αS amyloids bind to double-stranded DNA with micromolar affinity. Such DNA-amyloid interactions result in both single- and double-strand DNA breaks. In support of biological relevance, DNA damage was found to be increased in yeast cells expressing human αS that had formed amyloids. We propose that DNA damage represents a toxic gain-of-function chemical activity of αS amyloids that contributes to disease progression.

2. Results

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2.1. Amyloids of αS Bind DNA

To assess if αS amyloids can interact with double-stranded DNA (dsDNA), we employed surface plasmon resonance (SPR) analysis. αS in monomeric or amyloid form was injected in increasing concentrations over immobilized 50 base-pair, dsDNA molecules. The SPR response data show that αS amyloids (but not monomers) are capable of binding to the dsDNA at this condition (Figures 1A and S1). The apparent KD estimated for the αS amyloid-DNA interaction is 4.0 μM ± 2.1 μM (using αS concentration in monomer units). This observation suggests that amyloid-DNA interactions may also occur in vivo as intracellular αS concentrations are estimated to be in the 5–50 μM range. (40)

Figure 1

Figure 1. (A) Binding of monomeric (squares) and amyloid (circles) αS to immobilized DNA as measured by SPR, solid line shows hyperbolic fit. (B) AFM image of λ-DNA on mica surface. (C) AFM image of mixture of DNA and αS amyloids; blue arrows highlight where DNA appears to emerge after following along the amyloid long axis. Z-range for AFM images is 5 nm. (D) Box plot of height distribution of αS amyloids in the presence (average: 7.3 ± 1.0 nm) and absence (average: 6.1 ± 0.7 nm) of λ-DNA (P ≪ 0.0001). Inset shows an example cross section of λ-DNA (blue), αS amyloid alone (black) and αS amyloid with DNA (red).

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To visualize the amyloid–DNA interactions, we used AFM to analyze incubated mixtures of dsDNA (here, λ-DNA, 48.5 kbp) and αS amyloids that had been deposited on mica surfaces. DNA alone showed (as expected, (41)) elongated curly structures with heights of 0.6–0.8 nm (Figure 1B). For mixtures of DNA and αS amyloids, DNA molecules were always found in proximity to the αS amyloids. The DNA molecules appeared to run along the amyloid fibril long axis for extended distances and then protrude and reach over to other amyloid fibrils, creating a network of DNA-bridged amyloids (blue arrows in Figure 1C, additional images in Figure S2). The αS amyloids appeared similar by AFM as to without DNA (Figure S2) but their heights increased by approximately 1 nm in the presence of DNA (Figure 1D) in accordance with most amyloids being covered by one or two dsDNA molecules.

2.2. Amyloids of αS Damage DNA

To investigate if the interaction between αS amyloids and dsDNA results in chemical perturbation of the DNA, we first used a single molecule imaging technique to assesses single-strand damage. (42−44) After overnight incubation of αS amyloid and λ-DNA, the protein was removed and the formation of single-strand DNA lesions was probed with an enzyme cocktail of glycosylases and endonucleases (see Materials and Methods). These enzymes recognize different lesions (including oxidized bases, alkylated bases, nicks, abasic sites, and uracils) and then prepare the site for gap filling. (44,45) By subsequent addition of DNA polymerase 1, and a combination of unlabeled dNTPs and fluorescently labeled aminoallyl-dUTP-ATTO-647N, damaged sites are repaired (Figure 2A). Each repaired lesion becomes fluorescently labeled because the polymerase is progressive and inserts many bases around the damage site of which likely at least one will be a labeled UTP. To quantify the number of DNA damage sites, the DNA backbone was labeled by YOYO-1, a bis-intercalating fluorescent dye commonly used to stain DNA, (46) followed by stretching on silanized coverslips. The damage sites are observed as fluorescent “dots” along the stretched DNA molecules (Figure 2B). Upon quantifying detected damage sites on the DNA, we find a significant increase after incubation with αS amyloids as compared to dsDNA alone or upon incubation with αS monomers (Figure 2C). Similar results were obtained when amyloids of a C-terminally truncated αS form, αS(1–119), with 21 residues in the C-terminus removed, were incubated with DNA (Figure S3).

Figure 2

Figure 2. (A) Scheme of DNA damage detection. λ-DNA incubation with αS amyloids or αS monomers was followed by enzymatic repair and thereafter incorporation of fluorescent nucleotides at the damage sites. (B) Fluorescence microscopy image of labeled λ-DNA after incubation with αS monomers or amyloids and stretched on a functionalized glass coverslip. The DNA backbone was stained with YOYO-1 (green) and red dots are fluorescent nucleotides incorporated at damage sites. Scale bar = 10 μm. (C) DNA damage detection using a repair enzyme cocktail. Error bars indicate standard deviation calculated from biological replicates. (D) Detection of DNA damage using single repair enzymes. Error bars indicate standard deviation calculated from technical duplicates. P-values; ns, not significant; ***P ≤ 0.0002; ****P < 0.0001.

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To assess what type of dsDNA damage the αS amyloids promote, the repair enzyme cocktail constituents were assessed one by one. From the results of such experiments (Figure 2D), we found Endo IV and APE1 enzymes to be most active, suggesting that the types of lesions they repair are what the αS amyloids are mostly causing. Endo IV and APE1 have endonucleolytic and phosphoglycolate activities; they often repair nicked, abasic and oxidatively damaged sites. (47−51)
Notably, the enzymes in the cocktail do not probe dsDNA breaks. However, the analysis also reveals the length of the DNA molecules on the coverslips, which can hint to putative double-stranded breaks (observed as shorter DNA molecules). Such analysis indicated shorter dsDNA molecules after αS amyloid incubation (Figure S4), but for more accurate analysis of DNA length changes, we turned to nanochannel studies in solution.

2.3. Amyloids of αS Cleave DNA

To investigate if αS amyloids can cause DNA double-strand breaks, we assessed the length of individual λ-DNA molecules in the presence of αS amyloids using nanofluidics (Figure 3A). The length measurements were again facilitated using YOYO-1. Samples of 5 μM λ-DNA (base-pair) mixed with varying αS amyloid concentrations (0, 2.5, 4, and 10 μM) were analyzed (Figure 3B). The results from length measurements of ∼250 DNA molecules for each amyloid concentration (Figure 3C) reveal that the median length of individual DNA molecules reduced from 6.2 μm (control) to 5.1 μm (2.5 μM αS amyloid) to 4.5 μm (4 μM αS amyloid). There is a considerable increase in the presence of very small DNA molecules (length below 4 μm) when αS amyloids are present (27% for 2.5 μM, 43% for 4 μM and 37% for 10 μM αS amyloid) as compared to DNA alone (13% shorter than 4 μm).

Figure 3

Figure 3. (A) Schematic of the nanofluidic device. (B) Fluorescence images of λ-DNA molecules after incubation (and removal) with 0 μM (control, only DNA), 2.5, 4 and 10 μM αS amyloids in the nanochannels. (C) Distribution of lengths of λ-DNA molecules in the nanochannels. Median length of DNA molecules (arrows) and percentage of molecules with lengths of 4 μm or less are indicated in each panel.

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We exclude DNA compaction as the explanation for the observed shorter DNA for several reasons. First, if it had been DNA compaction, we would expect a gradual decrease in length for the whole population of DNA molecules. Notably, this is what was observed for the C-terminally truncated aS(1–97) monomer interacting with DNA (gradually shorter length for the DNA population, up to 25% length reduction, as a function of αS concentration) in an earlier study where DNA compaction was proposed. (30) Instead, we here observe a wide span of lengths with many very short DNA molecules (lengths reduced by up to 75%) in the presence of αS amyloid fibrils, while some molecules remain intact. Second, if the very short DNA molecules we observe had been compacted DNA, they should collapse fully to a blob, not stop at a short but still elongated state. (52) Many earlier studies have discussed how genomic DNA molecules adopt a toroidal shape when compacted in the presence of compaction agents. (53) Such blob-like conformations have been shown in earlier nanochannel studies involving for example heat-stable nucleoid-structuring protein (H-NS) and dextran as compacting agents. (54) Moreover, before complete compaction of a DNA molecule, the DNA is often partly compacted locally along the DNA, leading to varying dye emission intensity along the DNA due to variation in the local amount of DNA along the DNA contour. (55,56) We do not observe this uneven behavior for the longer (but still shortened) DNA molecules. Based on these observations, we exclude DNA compaction and conclude that reduced lengths of the DNA molecules in the presence of αS amyloids is due to DNA fragmentation. DNA fragmentation may be a result of direct double-strand or single-strand breaks on opposite DNA strands close enough to each other to allow the molecule to break into two pieces with single-stranded overhangs on each end.
When the αS amyloid concentration was 10 μM, we observed DNA lengths varying between 1 and 8.5 μm, i.e., in addition to many short molecules we also detected some that appear longer than the median DNA length in the control (DNA only) experiment. Since we previously found (using the same method) that wildtype αS monomers increase the length of DNA molecules, we speculate that there is a small fraction of αS monomers in our samples that cause the extended DNA molecules. When αS amyloid formation reaches saturation, fibrils are in equilibrium with monomers. Reported values of the equilibrium concentration of monomers range between 0.7 and 28 μM. (57) In our hands, typically 5–10% of the initial αS monomers remain as monomers after a completed aggregation experiment. Although we remove residual monomers before our experiments, the amyloids may shed some monomers during the incubation with DNA. At 10 μM αS amyloid concentration, the concentration of monomers in the sample may be sufficient to result in a detectable (but small) amount of extended DNA molecules.

2.4. Increased DNA Damage in αS Expressing Yeast

To assess αS-induced DNA damage in living cells, we turned to a yeast model system. In accord with the in vitro data, we find more DNA damage (detected by the double-stranded DNA break sensor protein Ddc2 labeled with GFP (58) Figure 4A) in actively growing cells expressing high levels of αS than in cells transformed with an empty vector. On average 14.1 ± 1.8% of control cells contained Ddc2 foci whereas 70.3 ± 4.3% of αS expressing cells contained Ddc2-GFP foci (Figure 4C). We confirmed the localization of these foci to the nuclei using the nucleolar/nuclear marker protein Sik1-RFP (Figure 4B).

Figure 4

Figure 4. Analysis of DNA damage in actively growing yeast cells. Exponentially growing cells expressing the double-stranded DNA break sensor protein Ddc2 fused to GFP (58) were imaged by fluorescence microscopy. (A) Cells were transformed with either the empty multicopy vector control plasmid (pYX242) or αS expressed from a strong, constitutive promotor. (B) To verify nuclear localization of Ddc2-GFP foci, cells were also transformed with a plasmid expressing a Sik1/Nop56-RFP fusion protein (71) and imaged by fluorescence microscopy. (C) Quantification of the fraction of control and αS expressing cells displaying Ddc2-GFP foci. On average 14.1 ± 1.8 (5.6% SD) of control cells contained foci whereas 70.3 ± 4.3 (16.1% SD) of αS expressing cells contained foci. A two-sided and two-tailed t-test (n = 10 vs n = 14) indicates a statistically significant difference with P < 4.7 × 10–10. (D) Cells expressing GFP tagged αS or GFP only (green) from a strong constitutive promoter were stained with Amytracker (red) to assess presence of amyloids.

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Thioflavin T positive inclusions have been shown in yeast that expresses αS at high levels. (59) In similarity, αS in our yeast system is expressed under the control of a strong constitutive promoter and from a multicopy plasmid. We directly confirmed the presence of amyloids in our yeast cells using the fluorescent amyloid-specific dye Amytracker in combination with GFP-tagged αS expression. The colocalization of Amytracker and αS signals inside the yeast cells confirms that the expressed αS indeed forms amyloids also in this model (Figure 4D).

3. Discussion

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Here we report that αS amyloids interact with dsDNA and that such interactions result in chemical modification of the DNA. The amyloid structure is required for activity as the presence of αS monomers does not result in detectable DNA modifications. From a biophysical perspective, this adds a new activity to the repertoire of chemical reactivity that is emerging for biological amyloid fibrils.
We recently reported catalytic activity of αS amyloids in vitro in the form of esterase and phosphatase activity on model ester and phosphoester substrates (35,36) (substrates shown in Figure 5B). Similar activities have been reported for amyloid-β and glucagon amyloids; in addition, dephosphorylation of ATP (also shown in Figure 5B) was reported for the latter amyloid. (38,39) Since the phosphate groups in the DNA backbone are exposed on dsDNA molecules, we speculate that αS amyloids damage DNA by phosphoester bond cleavage. If so, one expects αS amyloids, like glucagon amyloids, to hydrolyze ATP. Indeed, by the use of malachite green to detect inorganic phosphate, (60) we observed a buildup of free phosphate when αS amyloids were incubated with ATP (Figure S5).

Figure 5

Figure 5. (A) Illustration of possible amyloid-DNA interaction. High-resolution structure of wild-type αS amyloid (6h6b) with 5 layers of monomers in two protofilaments is shown next to a piece of B-form DNA (3bse) positioned at the suggested interaction site near the protofilament interface (see text). The surface of the αS amyloid is colored according to electrostatics (blue, positive; red, negative); in the DNA, phosphorus is orange and oxygen is red. The positions where N- and C-termini disordered segments will extend from the ordered amyloid core are indicated. (B) Chemical structures of substrates (PNPA, PNPP, ATP, DNA; the latter two, this work) reported to be cleaved by αS amyloids so far. PNPA, p-nitrophenyl acetate (ester bond); PNPP, p-nitrophenyl phosphate (phosphoester bond). Phosphodiester bonds, proposed cleavage sites in DNA, are marked with red arrows in the DNA chemical structure. We note that other bonds in the DNA backbone may also be targets for the amyloid reactivity.

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The αS amyloid structure has an ordered core with a repetitive surface pattern of identical residues running along the fiber long axis (Figure 5A). In surface cavities on the ordered amyloid structures, arrays of reactive sites may exist. Inspection of a typical wild-type αS amyloid structure (6h6b.pdb, there are several with similar overall fold) reveals a positive cleft with lysine residues running along the interface between the two protofilaments (Figure 5A, more structures in Figure S6). We propose that this region interacts with the negatively charged phosphates of the DNA backbone. When comparing dimensions, there will be one phosphate group of the twisting DNA helix directly facing the amyloid at every third (for minor groove) or fifth (for major groove) layer of the amyloid fibril (see Figure 5A, note that it is a hypothetical model only). Electrostatic attraction may pull on the DNA backbone toward the positively charged amyloid cleft. The affinity will be magnified due to the repetitive nature of both molecules and, at some places along the interface, protruding side chains on the amyloid surface may create sites that favor cleavage of DNA phosphodiester bonds (Figure 5B). Notably, the ordered αS amyloid core is surrounded by floppy N- (approximately 40 residues) and C-termini (approximately 45 residues) that do not adopt well-defined structures when analyzed by cryo-EM and other high-resolution methods (the positions of these extensions in the amyloid core are indicated in Figure 5A). Instead, they are thought to form a “fuzzy coat” surrounding the amyloid core. These peptides may affect interactions between the amyloid core and DNA; in fact, they could be responsible in full for the DNA interactions. Parts of the flexible N-terminus of αS harbor many positively charged side chains that may interact with DNA independently or provide stabilizing interactions around the DNA in addition to core amyloid interactions. In accord with termini contributions, another study showed that when a protein bound to the C-terminal floppy parts of αS amyloids, part of the αS floppy N-terminus folded onto the amyloid core. (61) We did not find increased DNA damage activity by αS amyloids with a C-terminal truncation of 21 residues, implying that the floppy C-terminus does not block DNA interactions. However, many further studies of reaction mechanisms and substrate binding sites are needed to understand how αS amyloids chemically damage DNA on a molecular level. From a fundamental scientific view, the range of chemical reactivity that is harbored in biological amyloids (that can adopt many polymorphs of the common cross-β structure) may be vast and deserves exploration.
Although most of our experiments involve purified αS and DNA in vitro, the findings have biological significance as αS amyloids are found in nuclei of neuronal cells, along with widespread DNA damage, in PD patients and animal models. (15,16) Many studies focus their investigations on Lewy body formation in the cytoplasm, but consistently report αS positive inclusions also in the nuclei. (32−34) One study could demonstrate, using GFP-tagged aS, that nuclear αS amyloids are able to move between cells and enter nuclei of cells not expressing GFP-tagged αS. (31) Since monomeric αS is present in the nucleus at normal conditions, (19) and moves in and out in a dynamic fashion, (21,22) amyloid formation may also be triggered directly in the nucleus from monomers residing there. Several in vitro studies have demonstrated that the presence of DNA can stimulate αS amyloid formation. (27,28) Even if DNA is wrapped around histones and interacts with other proteins in nuclei, the DNA is exposed at transcriptionally active sites. In accord with our findings, Vasquez et al. showed nuclear localization of αS to be necessary for genome damage in cultured neurons. (26)
From a clinical perspective, amyloid formation in neuronal cell nuclei in PD patients may be destructive in two ways: by sequestering αS monomers and thereby blocking their proposed DNA repair activities (17) (loss-of-function), as well as by inducing DNA chemical perturbation by direct DNA-amyloid interactions (toxic gain-of-function). αS amyloids may not only damage nuclear DNA: cytoplasmic αS amyloids may damage both RNA molecules and mitochondrial DNA, if mitochondrial membranes are perturbed. Indeed, mitochondrial dysfunction (which includes mitochondrial genome instability) is another signature of PD. (19,62) Our work suggests that in addition to several reported toxic effects of αS amyloids, αS amyloids may also contribute to disease progression by direct chemical damage of DNA.

4. Materials and Methods

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4.1. aS Expression and Purification

Wild-type and C-terminally truncated αS [αS(1–119); 21 C-terminal residues removed] was expressed in Escherichia coli grown in LB medium and purified using anion exchange chromatography and size exclusion chromatography as previously reported. (63) Purified protein was stored at −80 °C. Before each experiment, gel filtration was performed to obtain homogeneous monomeric αS using a Superdex 75 10/300 (Cytiva, Uppsala, Sweden) column in TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.6 at 25 °C, Medicago, Uppsala, Sweden).

4.2. Preparation of αS Amyloids

Amyloids of αS were prepared as described earlier. (36) In short, freshly gel filtered monomeric αS was incubated with ∼5% premade amyloid fibrils for 5 days at 37 °C. Following incubation, samples were centrifuged at 13,400 rpm for 30 min. The pellet was resuspended in TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). The amount of monomers that became amyloids were determined indirectly by measuring protein concentration left in the supernatant (as a measure of nonamyloid protein) using absorbance at 280 nm (extinction coefficient for αS of 5960 M–1 cm–1). In all experiments the concentration of amyloid fibrils denotes the monomer-equivalent concentration.

4.3. Surface Plasmon Resonance

Interactions between αS amyloids and DNA were studied using SPR on a Biacore X100 instrument with streptavidin coated sensor chip (Cytiva, Uppsala, Sweden). Amyloid fibrils of αS (prepared as above) were first sonicated to obtain shorter amyloids. Sonication was performed for 10 s using a probe sonicator (stepped microtip with Ultrasonic Processor Sonics Vibra-Cell; Sonics & Materials, Newtown, CT) running at 20% amplitude in an alternating cycle of 5 s (on mode) and 5 s (off mode). A 50-bp double-stranded biotin-labeled DNA (3′-CCTCTAGACCTGTACTACTCGAGAGATCGATCGACAGACGATGACTTAGC-5′) (Merck, Darmstadt, Germany) was immobilized on the sensor surface as described earlier. (64) The level of immobilization was 200 RU. Single cycle measurements were performed by injecting increasing concentrations up to 5 μM of monomeric or fibrillar αS on the surface. Five M NaCl was used to regenerate the surface after each cycle. Background correction was done by subtracting the signal of the flow channel where no DNA was immobilized. The running buffer was HBS-P (10 mM HEPES, 15 0 mM NaCl supplemented with 0.002% P20 detergent) (Cytiva, Uppsala, Sweden). The dissociation constant was obtained by fitting of the binding levels at the end of the injection versus protein concentration data to a 1:1 binding model (using αS monomer concentrations) using evaluation software provided by the manufacturer (Cytiva, Uppsala, Sweden). The dissociation constant obtained is an average of 3 independent experiments.

4.4. Atomic Force Microscopy (AFM)

Prior to imaging, 50 ng/μL of λ-DNA (48.5 kb, Thermo Fisher, Waltham, MA, USA) in the absence or presence of 40 μM αS amyloids as well as αS amyloid fibrils alone, were incubated overnight at room temperature in TE buffer. Deposition of DNA and protein samples on mica surface were performed according to published guidelines. (65) Freshly cleaved mica (Ted Pella Inc., Redding, CA, USA) was treated with 100 mM NiCl2 for 1 min and washed with Milli-Q grade water 3 times. The samples were diluted 10 times in 10 mM MgCl2, 25 mM KCl, 10 mM HEPES (pH 7.5) and incubated on the mica for 10 min followed by washing with Milli-Q grade water and drying with a gentle N2 flow. Images were recorded on an NTEGRA Prima setup (NT-MDT, Moscow, Russia) using a gold-coated single crystal silicon cantilever (NT-MDT, NSG01, spring constant of ∼5.1 N/m) and a resonance frequency of ∼180 kHz in tapping mode. 512 × 512-pixel images were acquired with a scan rate of 0.5 Hz. Images were analyzed using the WSxM 5.0 software. For the determination of αS amyloid heights, at least nine 5 × 5 μm images were taken in three different areas of the mica. The amyloid fibrils were automatically identified and average height of each individual fiber was measured using flooding analysis using the WSxM software. (66) The presented data is based on 160 amyloid fibers for each condition.

4.5. DNA Damage Assay Using Repair Enzymes

50 ng/μL of λ-DNA in the absence or presence of 40 μM αS amyloid fibrils or monomers were incubated in TE buffer overnight at room temperature. The DNA was separated from the amyloids using the Genomic DNA Clean and Concentrator-10 kit (D4010, Zymo research) before labeling of damage sites. For this, 100 ng of the purified λ-DNA was incubated with a cocktail of repair enzymes which consists of 2.5 U each of APE1, Endo III, Endo IV, Endo VIII, hAAG, Fpg, and UDG, in 1× CutSmart Buffer (New England BioLabs) for 1 h at 37 °C. This was followed by incubation with dNTPs (1 μM of dATP, dGTP, dCTP, 0.25 μM dTTP (Bionordika Sweden) and 0.25 μM aminoallyl-dUTP-ATTO-647N (Jena Bioscience)) in 1× NE Buffer 2 (Bionordika Sweden) and 1.25 U DNA polymerase 1 (Promega) for 1 h at 20 °C. The reaction was terminated with 2.5 μL of 0.25 M EDTA (Sigma-Aldrich). Samples were stored at −20 °C until imaged on chemically modified glass coverslips.
Glass coverslips (18 × 18 mm2) were arranged in a coverslip rack and immersed in an acetone solution containing 1% (3-aminopropyl)triethoxysilane and 1% allyltrimethoxysilane (Sigma-Aldrich). After activation, the coverslips were rinsed with a (2:1 v/v) acetone/water solution and dried using N2 gas flow prior to DNA sample addition. Prior to analysis, the DNA samples were stained with 320 nM YOYO-1 (Invitrogen) in 0.5× TBE, supplemented with 2% β-mercaptoethanol (BME, Sigma-Aldrich) to prevent photobleaching, in a final volume of 50 μL. Next the DNA samples were added to the coverslips. To stretch the DNA, 3.2 μL of stained DNA sample was placed at the interface of a silanized glass coverslip and a clean microscopy slide (VWR).
Imaging of stretched DNA molecules were performed using an epifluorescence microscope (Zeiss AxioObserver.Z1) equipped with a Colibri 7 LED light source. For the DNA damage assay, the microscope was equipped with an Andor iXON Ultra EMCCD camera and 100× oil immersion objective. Band-pass excitation filters (475/40 and 640/30 nm) and bandpass emission filters (530/50 and 690/50 nm) were used for YOYO-1 and aminoallyl-dUTP-ATTO-647, respectively.
Data was analyzed with custom-made MATLAB software. DNA molecules were detected by the software to measure DNA length and count colocalized aminoallyl-dUTP-ATTO-647N labels (dots) along the DNA. The results were expressed as dots/μm. This was then converted to dots per megabase pairs (dots/Mbp) using a conversion factor of 3000 bp/μm estimated from stretching of intact λ-DNA molecules. Dots at ends of molecules, which could result from breaks during sample handling, and overlapping molecules were excluded. Damage, expressed as dots/Mbp, thus corresponds to the total number of damage sites detected per Mbp DNA.
To assess statistical significance, experiments were performed in biological replicates (4 and 2 for amyloid and monomer experiments, respectively) unless otherwise noted, and differences between groups were assessed by one-way Anova with Tukey’s multiple comparison test, with a family wise alpha threshold and confidence level of 95% (confidence interval). Total number of images analyzed for λ-DNA, λ-DNA + αS monomers and λ-DNA + αS amyloids were 85, 37 and 67, respectively, and at least 7000 DNA molecules (corresponding to over 300 Mbp) in total were analyzed for each condition. P-values are represented using the GraphPad Prism style; ns, not significant; ***P ≤ 0.0002; ****P < 0.0001.

4.6. Nanofluidic DNA Length Experiments

Lengths of individual DNA molecules as a function of added αS amyloids were measured by confining DNA in nanofluidic channels. For this, 5 μM (base-pair) λ-DNA was incubated with varying concentrations (0, 2.5, 4 and 10 μM) of αS amyloids in 1 × TE (10 mM Tris and 1 mM EDTA) buffer at room temperature for 4 h. After the incubation, YOYO-1 dye was added to the samples at 1:5 dye to base pair ratio and incubated at room temperature for 30 min. 3% (v/v) BME was added as an oxygen scavenger to suppress oxygen radical induced photodamage of the DNA.
The nanofluidic devices were fabricated in a cleanroom facility using standard semiconductor fabrication procedures, the details of which are described in detail elsewhere. (67,68) Briefly, each device consists of two microfluidic channels that are 850 nm deep, and each microfluidic channel being connected to two sample loading reservoirs at its ends. The two microfluidic channels are connected by 200 parallel nanofluidic channels, with each nanofluidic channel being 150 nm in width, 100 nm in depth and 500 μm in length. The sample is loaded in one of the four loading reservoirs and the other three loading reservoirs are filled with buffer only. N2 pressure (2 bar) was applied to push the sample first from the loading reservoir into the microchannels and then into the nanochannels. DNA molecules are stretched in the nanofluidic channels due to nanoconfinement. For these experiments, the microscope described above was equipped with a Photometrics Evolve EMCCD camera, a 63× oil immersion objective and band-pass excitation (475/40 nm) and emission (530/50 nm) filters were used for YOYO-1 imaging. Using the imaging software ZEN, 20 subsequent images were recorded with an exposure time of 100 ms. Analysis of DNA lengths was performed using a custom-written MATLAB code after converting images to TIFF. Histogram plots (Figure 3C) were made using Origin Pro 2022b with X-axis as DNA length (μm) and Y-axis as number of DNA molecules (counts), with bin size of 0.3 μm.

4.7. DNA Damage in Yeast Cells

Saccharomyces cerevisiae yeast transformed with a multicopy plasmid expressing αS under the control a strong constitutive promoter (69) was used in parallel with an empty-vector control strain (transformed with the empty vector pYX242). The proportion of cells exhibiting nuclear foci of the double-stranded DNA break sensor protein Lcd1/Ddc2 was compared for yeast with and without aS. For this, cells expressing a genomic Lcd1/Ddc2 GFP fusion protein were employed. (58,70) Cells were grown overnight, diluted to OD 0.1, grown until in exponential phase (A600 = 1.2) and imaged using a Zeiss AxioObserver.Z1 inverted microscope equipped with Apotome/Axiocam 506 camera with a Plan-Apochromat 100x/1.40 Oil DIC M27 objective.
The percentage of cells containing Ddc2-GFP foci were evaluated in 16 different z-stacks per image. For empty vector yeast, a total of 854 cells were imaged in 3 independent experiments comprising in total 10 different images. For αS expressing cells, 1610 cells were imaged in 3 independent experiments comprising 14 different images. Cells were also transformed with the nucleolar/nuclear marker protein Sik1/Nop56-RFP (71) and grown to exponential phase (A600 = 1.2) in synthetic defined (SD) glucose medium lacking uracil. (70) Doubly labeled cells expressing αS were used to confirm nuclear localization (red, Nop56-RFP) of the Ddc2-GFP foci (green). To confirm amyloid formation of αS expressed in yeast, cells expressing αS-GFP or GFP only from a constitutive strong promoter (pRS426-GPD-αS-GFP or pRS426-GPD-GFP) (72) were grown to midexponential phase and stained with 1:100 diluted Amytracker 680 (1 mg/mL dissolved in DMSO, Ebba Biotech, Stockholm, Sweden) for 6 h before visualized in the fluorescence microscope.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.4c00461.

  • Figure S1 (SPR sensograms for binding), Figure S2 (additional AFM images of αS and DNA), Figure S3 [DNA damage induced by αS(1–119) amyloids], Figure S4 (DNA length distributions from analysis of DNA on coverslips), Figure S5 (ATPase activity for αS amyloids), and Figure S6 (high-resolution structures of the αS amyloid fold with putative DNA-binding site indicated) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Istvan Horvath - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
    • Obed Akwasi Aning - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
    • Sriram KK - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden;  https://orcid.org/0000-0002-4661-242X
    • Nikita Rehnberg - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden;  https://orcid.org/0009-0005-4879-1280
    • Srishti Chawla - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden
    • Mikael Molin - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden;  https://orcid.org/0000-0002-3903-8503
    • Fredrik Westerlund - Department of Life Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden;  https://orcid.org/0000-0002-4767-4868
  • Author Contributions

    Shared first authors. IH and PWS conceived the idea. IH, OA, SKK, NR, CS, MM designed and performed experiments. IH, PWS, OA, SKK, MM, FW analyzed data. IH, PWS, OA, SKK, FW wrote the manuscript. All authors edited the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Ranjeet Kumar and Brian Zhou for their experimental support. This work was funded by the Swedish Research Council (2023-03427 and 2019-03673 to PWS and 2020-03400 to FW), the Knut and Alice Wallenberg Foundation (Scholar grant to PWS), the European Research Council (Consolidator Grant, no. 866238 to FW), the Swedish Cancer Foundation (201145 PjF to FW), the Swedish Child Cancer Foundation (PR2022-0014 to FW) and the Wenner-Gren foundation (to OA). The Matlab software used to analyze single DNA molecule data was written by the group of T. Ambjörnsson (Lund University). The nanofluidic devices used in this study were fabricated at MyFab Chalmers cleanroom facility.

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    Goncalves, S.; Outeiro, T. F. Assessing the subcellular dynamics of alpha-synuclein using photoactivation microscopy. Mol. Neurobiol. 2013, 47 (3), 1081– 1092,  DOI: 10.1007/s12035-013-8406-x
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    Pinho, R.; Paiva, I.; Jercic, K. G.; Fonseca-Ornelas, L.; Gerhardt, E.; Fahlbusch, C. Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein. Hum. Mol. Genet. 2019, 28 (1), 31– 50,  DOI: 10.1093/hmg/ddy326
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    Kontopoulos, E.; Parvin, J. D.; Feany, M. B. Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity. Hum. Mol. Genet. 2006, 15 (20), 3012– 3023,  DOI: 10.1093/hmg/ddl243
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    Siddiqui, A.; Chinta, S. J.; Mallajosyula, J. K.; Rajagopolan, S.; Hanson, I.; Rane, A. Selective binding of nuclear alpha-synuclein to the PGC1alpha promoter under conditions of oxidative stress may contribute to losses in mitochondrial function: implications for Parkinson’s disease. Free Radical Biol. Med. 2012, 53 (4), 993– 1003,  DOI: 10.1016/j.freeradbiomed.2012.05.024
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    Vasquez, V.; Mitra, J.; Hegde, P. M.; Pandey, A.; Sengupta, S.; Mitra, S. Chromatin-Bound Oxidized alpha-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease. J. Alzheimer’s Dis. 2017, 60, S133– S150,  DOI: 10.3233/JAD-170342
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    Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson's Disease
    Vasquez, Velmarini; Mitra, Joy; Hegde, Pavana M.; Pandey, Arvind; Sengupta, Shiladitya; Mitra, Sankar; Rao, K. S.; Hegde, Muralidhar L.
    Journal of Alzheimer's Disease (2017), 60 (s1), S133-S150CODEN: JADIF9; ISSN:1387-2877. (IOS Press)
    Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathol. role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin assocn. of α-Syn in neurons using proximity ligation and chromatin immunopptn. anal. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus vs. cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chem. nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was obsd. in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel mol. insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.
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    Hegde, M. L.; Rao, K. S. DNA induces folding in alpha-synuclein: understanding the mechanism using chaperone property of osmolytes. Arch. Biochem. Biophys. 2007, 464 (1), 57– 69,  DOI: 10.1016/j.abb.2007.03.042
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    DNA induces folding in α-synuclein: Understanding the mechanism using chaperone property of osmolytes
    Hegde, Muralidhar L.; Rao, K. S. J.
    Archives of Biochemistry and Biophysics (2007), 464 (1), 57-69CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)
    α-Synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson's disease (PD). Previously, we have shown that α-synuclein has DNA binding activity. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of α-synuclein. It was obsd. that single-stranded circular DNA induces α-helix formation in α-synuclein while plasmid supercoiled DNA has dual effect, inducing a partially folded conformation and α-helix formation under different exptl. conditions. Interestingly, α-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced a partially folded conformation in α-synuclein promoted fibrillation, while DNA which induced α-helix formation delayed the fibrillation. This finding indicates that the partially folded intermediate conformation is crit. in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of α-synuclein was studied using the effect of osmolytes on α-synuclein as a model system. Among the five osmolytes used, glycerol, trimethylamine-N-oxide, betaine, and taurine induced a partially folded α-synuclein conformation and in turn enhanced the aggregation of α-synuclein. The ability of DNA and osmolytes to induce conformational transitions in α-synuclein indicates that the following two factors are crit. in modulating α-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The ability of DNA to induce α-helix formation in α-synuclein and inhibit fibrillation may be significant for engineering DNA-chip-based therapeutic approaches to PD and other amyloid disorders.
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    Cherny, D.; Hoyer, W.; Subramaniam, V.; Jovin, T. M. Double-stranded DNA stimulates the fibrillation of alpha-synuclein in vitro and is associated with the mature fibrils: an electron microscopy study. J. Mol. Biol. 2004, 344 (4), 929– 938,  DOI: 10.1016/j.jmb.2004.09.096
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    Double-stranded DNA Stimulates the Fibrillation of α-Synuclein in vitro and is Associated with the Mature Fibrils: An Electron Microscopy Study
    Cherny, Dmitry; Hoyer, Wolfgang; Subramaniam, Vinod; Jovin, Thomas M.
    Journal of Molecular Biology (2004), 344 (4), 929-938CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
    Filamentous aggregates formed by α-synuclein are a prominent and presumably key etiol. factor in Parkinson's and other neurodegenerative diseases characterized by motor disorders. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation properties of α-synuclein, e.g. by accelerating the process of assembly. Histones, the major component and constituent of chromatin, interact specifically with α-synuclein and enhance its fibrillation significantly. Here, we report that another component of chromatin, double-stranded DNA (dsDNA), either linear or supercoiled, also interacts with wild-type α-synuclein, leading to a significant stimulation of α-synuclein assembly into mature fibrils characterized by a reduced lag phase. In general, the morphol. of the fibrils remains unchanged in the presence of linear dsDNA. Electron microscopy reveals that DNA forms various types of complexes upon assocn. with the fibrils at their surface without distortion of the double-helical structure. The existence of these complexes was confirmed by the electrophoresis, which also demonstrated that a fraction of the assocd. DNA was resistant to digestion by restriction endonucleases. Fibrils assembled from the α-synuclein mutants A30P and A53T and the C-terminally truncated variants (encoding amino acid residues 1-108 or 1-124) also form complexes with linear dsDNA. Possible mechanisms and implications of dsDNA-α-synuclein interactions are discussed.
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    Jiang, K.; Rocha, S.; Westling, A.; Kesarimangalam, S.; Dorfman, K. D.; Wittung-Stafshede, P.; Westerlund, F. Alpha-Synuclein Modulates the Physical Properties of DNA. Chemistry 2018, 24 (58), 15685– 15690,  DOI: 10.1002/chem.201803933
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    Jiang, K.; Rocha, S.; Kumar, R.; Westerlund, F.; Wittung-Stafshede, P. C-terminal truncation of alpha-synuclein alters DNA structure from extension to compaction. Biochem. Biophys. Res. Commun. 2021, 568, 43– 47,  DOI: 10.1016/j.bbrc.2021.06.059
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    Weston, L. J.; Bowman, A. M.; Osterberg, V. R.; Meshul, C. K.; Woltjer, R. L.; Unni, V. K. Aggregated Alpha-Synuclein Inclusions within the Nucleus Predict Impending Neuronal Cell Death in a Mouse Model of Parkinsonism. Int. J. Mol. Sci. 2022, 23 (23), 15294,  DOI: 10.3390/ijms232315294
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    Koss, D. J.; Erskine, D.; Porter, A.; Palmoski, P.; Menon, H.; Todd, O. G. J.; Leite, M.; Attems, J.; Outeiro, T. F. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies. Acta Neuropathol. Commun. 2022, 10, 98,  DOI: 10.1186/s40478-022-01403-x
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    Wittung-Stafshede, P. Chemical catalysis by biological amyloids. Biochem. Soc. Trans. 2023, 51 (5), 1967– 1974,  DOI: 10.1042/BST20230617
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    Horvath, I.; Wittung-Stafshede, P. Amyloid Fibers of α-Synuclein Catalyze Chemical Reactions. ACS Chem. Neurosci. 2023, 14, 603– 608,  DOI: 10.1021/acschemneuro.2c00799
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    Horvath, I.; Mohamed, K. A.; Kumar, R.; Wittung-Stafshede, P. Amyloids of alpha-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites. Int. J. Mol. Sci. 2023, 24 (16), 12849,  DOI: 10.3390/ijms241612849
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    Arad, E.; Baruch Leshem, A.; Rapaport, H.; Jelinek, R. β-Amyloid fibrils catalyze neurotransmitter degradation. Chem Catal. 2021, 1 (4), 908– 922,  DOI: 10.1016/j.checat.2021.07.005
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    β-Amyloid fibrils catalyze neurotransmitter degradation
    Arad, Elad; Baruch Leshem, Avigail; Rapaport, Hanna; Jelinek, Raz
    Chem Catalysis (2021), 1 (4), 908-922CODEN: CCHAE9; ISSN:2667-1093. (Elsevier Inc.)
    Amyloid fibrils are one of the hallmarks of Alzheimer's disease (AD), although a causative link between plaque-forming amyloid fibrils and AD pathol. remains to be clarified. This study demonstrates, for the first time for a naturally occurring amyloid, that fibrils comprising the 42-residue amyloid-β peptide (Aβ42) exhibit significant catalytic properties. Aβ42 fibrils catalyzed the hydrolysis of the model ester para-nitrophenyl acetate (pNPA) and of acetylthiocholine, a surrogate for the neurotransmitter acetylcholine. Aβ42 fibrils also catalyzed oxidn. of the prominent neurotransmitters dopamine and adrenaline. Importantly, the catalytic activity was specifically manifested by mature Aβ42 fibrils and not the peptide monomers or oligomeric Aβ42, the putative neurotoxic species. Furthermore, maximal catalytic activity was recorded by the full-length Aβ42 fibrils, whereas fibrillar assemblies comprising Aβ42 subdomains were significantly less catalytic. The catalytic activity of Aβ fibrils could exhibit insidious roles in AD pathophysiol.
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    Arad, E.; Yosefi, G.; Kolusheva, S.; Bitton, R.; Rapaport, H.; Jelinek, R. Native Glucagon Amyloids Catalyze Key Metabolic Reactions. ACS Nano 2022, 16 (8), 12889– 12899,  DOI: 10.1021/acsnano.2c05166
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    Native Glucagon Amyloids Catalyze Key Metabolic Reactions
    Arad, Elad; Yosefi, Gal; Kolusheva, Sofiya; Bitton, Ronit; Rapaport, Hanna; Jelinek, Raz
    ACS Nano (2022), 16 (8), 12889-12899CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Glucagon is a prominent peptide hormone, playing central roles in the regulation of glucose blood-level and lipid metab. Formation of glucagon amyloid fibrils was previously reported, although no biol. functions of such fibrils are known. Here, glucagon amyloid fibrils catalyze biol. important reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. In particular, glucagon fibrils catalyze dephosphorylation of ATP, a core metabolic reaction in cell biol. Comparative anal. of several glucagon variants allowed mapping the catalytic activity to an enzymic pocket-like triad formed at the glucagon fibril surface, comprising the histidyl-serine domain at the N-terminus of the peptide. This study may point to previously unknown physiol. roles and pathol. consequences of glucagon fibrillation, and supports the hypothesis that catalytic activities of native amyloid fibrils play functional roles in human physiol. and disease.
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    Theillet, F.-X.; Binolfi, A.; Bekei, B.; Martorana, A.; Rose, H. M.; Stuiver, M. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016, 530, 45,  DOI: 10.1038/nature16531
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    Structural disorder of monomeric α-synuclein persists in mammalian cells
    Theillet, Francois-Xavier; Binolfi, Andres; Bekei, Beata; Martorana, Andrea; Rose, Honor May; Stuiver, Marchel; Verzini, Silvia; Lorenz, Dorothea; van Rossum, Marleen; Goldfarb, Daniella; Selenko, Philipp
    Nature (London, United Kingdom) (2016), 530 (7588), 45-50CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Intracellular aggregation of the human amyloid protein, α-synuclein (I), is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here, the authors used NMR and ESR spectroscopy to derive at.-resoln. insights into the structure and dynamics of I in different mammalian cell types. The authors showed that the disordered nature of monomeric I was stably preserved in non-neuronal and neuronal cells. Under physiol. cell conditions, I was N-terminally acetylated and adopted conformations that were more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results established that different types of crowded intracellular environments do not inherently promote I oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.
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    Cervantes, N. A. G.; Medina, B. G. Robust deposition of lambda DNA on mica for imaging by AFM in air. Scanning 2014, 36 (6), 561– 569,  DOI: 10.1002/sca.21155
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    Singh, V.; Johansson, P.; Ekedahl, E.; Lin, Y. L.; Hammarsten, O.; Westerlund, F. Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging. Biochem. Biophys. Res. Commun. 2022, 594, 57– 62,  DOI: 10.1016/j.bbrc.2022.01.041
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    Singh, V.; Johansson, P.; Lin, Y. L.; Hammarsten, O.; Westerlund, F. Shining light on single-strand lesions caused by the chemotherapy drug bleomycin. DNA Repair 2021, 105, 103153,  DOI: 10.1016/j.dnarep.2021.103153
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    Zirkin, S.; Fishman, S.; Sharim, H.; Michaeli, Y.; Don, J.; Ebenstein, Y. Lighting up individual DNA damage sites by in vitro repair synthesis. J. Am. Chem. Soc. 2014, 136 (21), 7771– 7776,  DOI: 10.1021/ja503677n
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    Lighting Up Individual DNA Damage Sites by In Vitro Repair Synthesis
    Zirkin, Shahar; Fishman, Sivan; Sharim, Hila; Michaeli, Yael; Don, Jeremy; Ebenstein, Yuval
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    DNA damage and repair are linked to fundamental biol. processes such as metab., disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-mol. imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quant. follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradn. in several cell types. Finally, we show the modularity of this single-mol. approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extd. from mouse brain tissue.
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    Boiteux, S.; Guillet, M. Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae. DNA Repair 2004, 3 (1), 1– 12,  DOI: 10.1016/j.dnarep.2003.10.002
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    Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae
    Boiteux, Serge; Guillet, Marie
    DNA Repair (2004), 3 (1), 1-12CODEN: DRNEAR; ISSN:1568-7864. (Elsevier Science B.V.)
    A review. Apurinic/apyrimidinic (AP) sites are one of the most frequent spontaneous lesions in DNA. They are potentially mutagenic and lethal lesions that can block DNA replication and transcription. In addn., cleavage of AP sites by AP endonucleases or AP lyases generates DNA single-strand breaks (SSBs) with 5'- or 3'-blocked ends, resp. Therefore, we suggest that AP sites and 3'- or 5'-blocked SSBs, we name "honorary AP sites", constitute a single class of lesions. In this review, we describe the different mechanisms used by the budding yeast Saccharomyces cerevisiae to remove or tolerate AP sites and related SSBs. In wild-type cells, AP sites are primarily repaired by the base excision repair (BER) pathway, with the nucleotide excision repair (NER) pathway as a back up activity. BER is initiated by one of the two AP endonucleases, Apn1 or Apn2. Three DNA N-glycosylases/AP lyases, Ntg1, Ntg2 and Ogg1, can also incise AP sites in DNA. Rad27, a structure specific endonuclease, is involved in the repair of 5'-blocked ends, whereas Apn1, Apn2 and Rad1-Rad10 are involved in the removal of 3'-blocked ends using their 3'-phosphodiesterase and 3'-flap endonuclease activities, resp. AP sites can stall DNA replication forks, as well as they block in vitro DNA synthesis by DNA polymerase δ. Restart of stalled forks can occur through a recombination-assocd. pathway initiated by the Mus81-Mms4 endonuclease or mutagenic translesion DNA synthesis (TLS). The mutagenic bypass of AP sites is a two-polymerases affair with an inserter DNA polymerase (Polδ, Polη or Rev1) and an extender DNA polymerase (Polζ). Under normal growth conditions, inactivation of Apn1, Apn2 and Rad1-Rad10 causes cell death. Therefore, the burden of spontaneous AP sites is not compatible with life, in the absence of excision repair pathways. These results in yeast demonstrate that AP sites are crit. endogenous DNA damages that cause genetic instability and by analogy could be assocd. with degenerative pathologies in human.
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    APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions
    Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P.; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K.
    Experimental & Molecular Medicine (2014), 46 (7), e106CODEN: EMMEF3; ISSN:2092-6413. (NPG Nature Asia-Pacific)
    A review. Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiol. processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metab., cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addn., studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.
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    A review. Compaction is the process in which a large DNA mol. undergoes a transition between an elongated conformation and a very compact form. In nature, DNA compaction occurs to package genomic material inside tiny spaces such as viral capsids and cell nuclei. In vitro, several strategies exist to compact DNA. In this review, we first provide a physico-chem. description of this phenomenon, focusing on the modes of compaction, the types of compaction agents and the chem. and phys. parameters that control compaction and its reverse process, decompaction. We then describe three main kinds of applications. First, we show how regulated compaction/decompaction can be used to control gene activity in vitro, with a particular emphasis on the use of light to reversibly control gene expression. Second, we describe several approaches where compaction is used as a way to reversibly protect DNA against chem., biochem., or mech. stresses. Third, we show that compact DNA can be used as a nanostructure template to generate nanomaterials with a well-defined size and shape. We conclude by proposing some perspectives for future biochem. and biotechnol. applications and enumerate some remaining challenges that we think worth being undertaken.
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  1. Claudio Castillo-Cáceres, Esteban Nova, Rodrigo Diaz-Espinoza. Alpha-synuclein amyloids catalyze the degradation of ATP and other nucleotides. Scientific Reports 2026, 16 (1) https://doi.org/10.1038/s41598-025-32888-w
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  • Abstract

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    Figure 1

    Figure 1. (A) Binding of monomeric (squares) and amyloid (circles) αS to immobilized DNA as measured by SPR, solid line shows hyperbolic fit. (B) AFM image of λ-DNA on mica surface. (C) AFM image of mixture of DNA and αS amyloids; blue arrows highlight where DNA appears to emerge after following along the amyloid long axis. Z-range for AFM images is 5 nm. (D) Box plot of height distribution of αS amyloids in the presence (average: 7.3 ± 1.0 nm) and absence (average: 6.1 ± 0.7 nm) of λ-DNA (P ≪ 0.0001). Inset shows an example cross section of λ-DNA (blue), αS amyloid alone (black) and αS amyloid with DNA (red).

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    Figure 2

    Figure 2. (A) Scheme of DNA damage detection. λ-DNA incubation with αS amyloids or αS monomers was followed by enzymatic repair and thereafter incorporation of fluorescent nucleotides at the damage sites. (B) Fluorescence microscopy image of labeled λ-DNA after incubation with αS monomers or amyloids and stretched on a functionalized glass coverslip. The DNA backbone was stained with YOYO-1 (green) and red dots are fluorescent nucleotides incorporated at damage sites. Scale bar = 10 μm. (C) DNA damage detection using a repair enzyme cocktail. Error bars indicate standard deviation calculated from biological replicates. (D) Detection of DNA damage using single repair enzymes. Error bars indicate standard deviation calculated from technical duplicates. P-values; ns, not significant; ***P ≤ 0.0002; ****P < 0.0001.

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    Figure 3

    Figure 3. (A) Schematic of the nanofluidic device. (B) Fluorescence images of λ-DNA molecules after incubation (and removal) with 0 μM (control, only DNA), 2.5, 4 and 10 μM αS amyloids in the nanochannels. (C) Distribution of lengths of λ-DNA molecules in the nanochannels. Median length of DNA molecules (arrows) and percentage of molecules with lengths of 4 μm or less are indicated in each panel.

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    Figure 4

    Figure 4. Analysis of DNA damage in actively growing yeast cells. Exponentially growing cells expressing the double-stranded DNA break sensor protein Ddc2 fused to GFP (58) were imaged by fluorescence microscopy. (A) Cells were transformed with either the empty multicopy vector control plasmid (pYX242) or αS expressed from a strong, constitutive promotor. (B) To verify nuclear localization of Ddc2-GFP foci, cells were also transformed with a plasmid expressing a Sik1/Nop56-RFP fusion protein (71) and imaged by fluorescence microscopy. (C) Quantification of the fraction of control and αS expressing cells displaying Ddc2-GFP foci. On average 14.1 ± 1.8 (5.6% SD) of control cells contained foci whereas 70.3 ± 4.3 (16.1% SD) of αS expressing cells contained foci. A two-sided and two-tailed t-test (n = 10 vs n = 14) indicates a statistically significant difference with P < 4.7 × 10–10. (D) Cells expressing GFP tagged αS or GFP only (green) from a strong constitutive promoter were stained with Amytracker (red) to assess presence of amyloids.

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    Figure 5

    Figure 5. (A) Illustration of possible amyloid-DNA interaction. High-resolution structure of wild-type αS amyloid (6h6b) with 5 layers of monomers in two protofilaments is shown next to a piece of B-form DNA (3bse) positioned at the suggested interaction site near the protofilament interface (see text). The surface of the αS amyloid is colored according to electrostatics (blue, positive; red, negative); in the DNA, phosphorus is orange and oxygen is red. The positions where N- and C-termini disordered segments will extend from the ordered amyloid core are indicated. (B) Chemical structures of substrates (PNPA, PNPP, ATP, DNA; the latter two, this work) reported to be cleaved by αS amyloids so far. PNPA, p-nitrophenyl acetate (ester bond); PNPP, p-nitrophenyl phosphate (phosphoester bond). Phosphodiester bonds, proposed cleavage sites in DNA, are marked with red arrows in the DNA chemical structure. We note that other bonds in the DNA backbone may also be targets for the amyloid reactivity.

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      Pathobiology of the Lewy body
      Galvin, James E.; Lee, Virginia M-Y.; Schmidt, M. Luise; Tu, Pan-Hsien; Iwatsubo, Takeshi; Trojanowski, John Q.
      Advances in Neurology (1999), 80 (Parkinson's Disease), 313-324CODEN: ADNRA3; ISSN:0091-3952. (Lippincott-Raven Publishers)
      A review, with 114 refs. Topics discussed include: characterization of Lewy body components in situ immunohistochem., monoclonal antibodies to purified Lewy bodies, pathol. models of Parkinson's disease and other Lewy body disorders, and transgenic mouse models that form Lewy body-like inclusions.
    10. 10
      Alam, Z. I.; Jenner, A.; Daniel, S. E.; Lees, A. J.; Cairns, N.; Marsden, C. D. Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J. Neurochem. 1997, 69 (3), 1196– 1203,  DOI: 10.1046/j.1471-4159.1997.69031196.x
      10
      Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra
      Alam, Z. I.; Jenner, A.; Daniel, S. E.; Lees, A. J.; Cairns, N.; Marsden, C. D.; Jenner, P.; Halliwell, B.
      Journal of Neurochemistry (1997), 69 (3), 1196-1203CODEN: JONRA9; ISSN:0022-3042. (Lippincott-Raven)
      Oxidative damage has been implicated in the pathol. of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatog./mass spectrometry was used to examine products of oxidn. and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examd., both normal and PD. Anal. showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra; rises in other base oxidn./deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO-) or hydroxyl radicals (OH.), or to be a prooxidant effect of treatment with L-Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.
    11. 11
      Kikuchi, A.; Takeda, A.; Onodera, H.; Kimpara, T.; Hisanaga, K.; Sato, N. Systemic increase of oxidative nucleic acid damage in Parkinson’s disease and multiple system atrophy. Neurobiol. Dis. 2002, 9 (2), 244– 248,  DOI: 10.1006/nbdi.2002.0466
      11
      Systemic Increase of Oxidative Nucleic Acid Damage in Parkinson's Disease and Multiple System Atrophy
      Kikuchi, Akio; Takeda, Atsushi; Onodera, Hiroshi; Kimpara, Teiko; Hisanaga, Kinya; Sato, Nobuyuki; Nunomura, Akihiko; Castellani, Rudy J.; Perry, George; Smith, Mark A.; Itoyama, Yasuto
      Neurobiology of Disease (2002), 9 (2), 244-248CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Science)
      8-Hydroxy-2'-deoxyguanosine (8-OHdG) or 8-hydroxyguanosine (8-OHG), a product of oxidized DNA or RNA, is a good marker of oxidative cellular damage. In this study, we measured the 8-OHdG/8-OHG levels in the serum and cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) and multiple system atrophy (MSA). Compared to age-matched controls, the mean levels of serum 8-OHdG/8-OHG were significantly higher in PD (P < 0.0001). Although no gender differences were obsd. in the controls, the mean values of serum 8-OHdG/8-OHG were significantly higher in female PD cases (P < 0.005) than in male patients. 8-OHdG/8-OHG levels in CSF were also increased significantly in patients with PD and MSA, however, their relative values were generally much lower than those in the serum. Together with previous studies showing increased peripheral 8-OHdG levels in Alzheimer's disease and amyotrophic lateral sclerosis, the data presented here suggest that systemic DNA/RNA oxidn. is commonly obsd. in neurodegenerative diseases. Our results also imply that female patients with PD show higher levels of oxidative stress, which may explain the faster progression of this disease in females.
    12. 12
      Li, Y. L.; Wang, Z. X.; Ying, C. Z.; Zhang, B. R.; Pu, J. L. Decoding the Role of Familial Parkinson’s Disease-Related Genes in DNA Damage and Repair. Aging Dis. 2022, 13 (5), 1405– 1412,  DOI: 10.14336/AD.2022.0216
      There is no corresponding record for this reference.
    13. 13
      López-Otín, C.; Blasco, M. A.; Partridge, L.; Serrano, M.; Kroemer, G. Hallmarks of aging: An expanding universe. Cell 2023, 186 (2), 243– 278,  DOI: 10.1016/j.cell.2022.11.001
      13
      Hallmarks of aging: An expanding universe
      Lopez-Otin, Carlos; Blasco, Maria A.; Partridge, Linda; Serrano, Manuel; Kroemer, Guido
      Cell (Cambridge, MA, United States) (2023), 186 (2), 243-278CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review. Aging is driven by hallmarks fulfilling the following three premises: (1) their age-assocd. manifestation, (2) the acceleration of aging by exptl. accentuating them, and (3) the opportunity to decelerate, stop, or reverse aging by therapeutic interventions on them. We propose the following twelve hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These hallmarks are interconnected among each other, as well as to the recently proposed hallmarks of health, which include organizational features of spatial compartmentalization, maintenance of homeostasis, and adequate responses to stress.
    14. 14
      Hegde, M. L.; Gupta, V. B.; Anitha, M.; Harikrishna, T.; Shankar, S. K.; Muthane, U. Studies on genomic DNA topology and stability in brain regions of Parkinson’s disease. Arch. Biochem. Biophys. 2006, 449 (1–2), 143– 156,  DOI: 10.1016/j.abb.2006.02.018
      There is no corresponding record for this reference.
    15. 15
      Coppedè, F.; Migliore, L. DNA damage in neurodegenerative diseases. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2015, 776, 84– 97,  DOI: 10.1016/j.mrfmmm.2014.11.010
      There is no corresponding record for this reference.
    16. 16
      Madabhushi, R.; Pan, L.; Tsai, L.-H. DNA Damage and Its Links to Neurodegeneration. Neuron 2014, 83 (2), 266– 282,  DOI: 10.1016/j.neuron.2014.06.034
      16
      DNA Damage and Its Links to Neurodegeneration
      Madabhushi, Ram; Pan, Ling; Tsai, Li-Huei
      Neuron (2014), 83 (2), 266-282CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      A review. The integrity of our genetic material is under const. attack from numerous endogenous and exogenous agents. The consequences of a defective DNA damage response are well studied in proliferating cells, esp. with regards to the development of cancer, yet its precise roles in the nervous system are relatively poorly understood. Here we attempt to provide a comprehensive overview of the consequences of genomic instability in the nervous system. We highlight the neuropathol. of congenital syndromes that result from mutations in DNA repair factors and underscore the importance of the DNA damage response in neural development. In addn., we describe the findings of recent studies, which reveal that a robust DNA damage response is also intimately connected to aging and the manifestation of age-related neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis.
    17. 17
      Schaser, A. J.; Osterberg, V. R.; Dent, S. E.; Stackhouse, T. L.; Wakeham, C. M.; Boutros, S. W.; Weston, L. J.; Owen, N.; Weissman, T. A.; Luna, E. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders. Sci. Rep. 2019, 9 (1), 10919,  DOI: 10.1038/s41598-019-47227-z
      17
      Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders
      Schaser Allison J; Osterberg Valerie R; Dent Sydney E; Stackhouse Teresa L; Weston Leah J; Unni Vivek K; Wakeham Colin M; Boutros Sydney W; Raber Jacob; Owen Nichole; McCullough Amanda K; Weissman Tamily A; Luna Esteban; Luk Kelvin C; McCullough Amanda K; Woltjer Randall L; Unni Vivek K
      Scientific reports (2019), 9 (1), 10919 ISSN:.
      Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.
    18. 18
      Chen, V.; Moncalvo, M.; Tringali, D.; Tagliafierro, L.; Shriskanda, A.; Ilich, E. The mechanistic role of alpha-synuclein in the nucleus: impaired nuclear function caused by familial Parkinson’s disease SNCA mutations. Hum. Mol. Genet. 2020, 29 (18), 3107– 3121,  DOI: 10.1093/hmg/ddaa183
      There is no corresponding record for this reference.
    19. 19
      Gonzalez-Hunt, C. P.; Sanders, L. H. DNA damage and repair in Parkinson’s disease: Recent advances and new opportunities. J. Neurosci. Res. 2021, 99 (1), 180– 189,  DOI: 10.1002/jnr.24592
      There is no corresponding record for this reference.
    20. 20
      Sampaio-Marques, B.; Guedes, A.; Vasilevskiy, I.; Gonçalves, S.; Outeiro, T. F.; Winderickx, J.; Burhans, W. C.; Ludovico, P. α-Synuclein toxicity in yeast and human cells is caused by cell cycle re-entry and autophagy degradation of ribonucleotide reductase 1. Aging Cell 2019, 18 (4), e12922  DOI: 10.1111/acel.12922
      There is no corresponding record for this reference.
    21. 21
      Goers, J.; Manning-Bog, A. B.; McCormack, A. L.; Millett, I. S.; Doniach, S.; Di Monte, D. A. Nuclear localization of alpha-synuclein and its interaction with histones. Biochemistry 2003, 42 (28), 8465– 8471,  DOI: 10.1021/bi0341152
      21
      Nuclear Localization of α-Synuclein and Its Interaction with Histones
      Goers, John; Manning-Bog, Amy B.; McCormack, Alison L.; Millett, Ian S.; Doniach, Sebastian; Di Monte, Donato A.; Uversky, Vladimir N.; Fink, Anthony L.
      Biochemistry (2003), 42 (28), 8465-8471CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The aggregation of α-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of α-synuclein under physiol. and pathol. conditions is unknown, and the mechanism of α-synuclein aggregation is not well understood. Here the authors show that α-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of α-synuclein is dramatically accelerated in the presence of histones in vitro. The authors also describe the presence of α-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying α-synuclein pathophysiol.
    22. 22
      Goncalves, S.; Outeiro, T. F. Assessing the subcellular dynamics of alpha-synuclein using photoactivation microscopy. Mol. Neurobiol. 2013, 47 (3), 1081– 1092,  DOI: 10.1007/s12035-013-8406-x
      There is no corresponding record for this reference.
    23. 23
      Pinho, R.; Paiva, I.; Jercic, K. G.; Fonseca-Ornelas, L.; Gerhardt, E.; Fahlbusch, C. Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein. Hum. Mol. Genet. 2019, 28 (1), 31– 50,  DOI: 10.1093/hmg/ddy326
      There is no corresponding record for this reference.
    24. 24
      Kontopoulos, E.; Parvin, J. D.; Feany, M. B. Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity. Hum. Mol. Genet. 2006, 15 (20), 3012– 3023,  DOI: 10.1093/hmg/ddl243
      There is no corresponding record for this reference.
    25. 25
      Siddiqui, A.; Chinta, S. J.; Mallajosyula, J. K.; Rajagopolan, S.; Hanson, I.; Rane, A. Selective binding of nuclear alpha-synuclein to the PGC1alpha promoter under conditions of oxidative stress may contribute to losses in mitochondrial function: implications for Parkinson’s disease. Free Radical Biol. Med. 2012, 53 (4), 993– 1003,  DOI: 10.1016/j.freeradbiomed.2012.05.024
      There is no corresponding record for this reference.
    26. 26
      Vasquez, V.; Mitra, J.; Hegde, P. M.; Pandey, A.; Sengupta, S.; Mitra, S. Chromatin-Bound Oxidized alpha-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease. J. Alzheimer’s Dis. 2017, 60, S133– S150,  DOI: 10.3233/JAD-170342
      26
      Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson's Disease
      Vasquez, Velmarini; Mitra, Joy; Hegde, Pavana M.; Pandey, Arvind; Sengupta, Shiladitya; Mitra, Sankar; Rao, K. S.; Hegde, Muralidhar L.
      Journal of Alzheimer's Disease (2017), 60 (s1), S133-S150CODEN: JADIF9; ISSN:1387-2877. (IOS Press)
      Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathol. role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin assocn. of α-Syn in neurons using proximity ligation and chromatin immunopptn. anal. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus vs. cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chem. nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was obsd. in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel mol. insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.
    27. 27
      Hegde, M. L.; Rao, K. S. DNA induces folding in alpha-synuclein: understanding the mechanism using chaperone property of osmolytes. Arch. Biochem. Biophys. 2007, 464 (1), 57– 69,  DOI: 10.1016/j.abb.2007.03.042
      27
      DNA induces folding in α-synuclein: Understanding the mechanism using chaperone property of osmolytes
      Hegde, Muralidhar L.; Rao, K. S. J.
      Archives of Biochemistry and Biophysics (2007), 464 (1), 57-69CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)
      α-Synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson's disease (PD). Previously, we have shown that α-synuclein has DNA binding activity. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of α-synuclein. It was obsd. that single-stranded circular DNA induces α-helix formation in α-synuclein while plasmid supercoiled DNA has dual effect, inducing a partially folded conformation and α-helix formation under different exptl. conditions. Interestingly, α-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced a partially folded conformation in α-synuclein promoted fibrillation, while DNA which induced α-helix formation delayed the fibrillation. This finding indicates that the partially folded intermediate conformation is crit. in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of α-synuclein was studied using the effect of osmolytes on α-synuclein as a model system. Among the five osmolytes used, glycerol, trimethylamine-N-oxide, betaine, and taurine induced a partially folded α-synuclein conformation and in turn enhanced the aggregation of α-synuclein. The ability of DNA and osmolytes to induce conformational transitions in α-synuclein indicates that the following two factors are crit. in modulating α-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The ability of DNA to induce α-helix formation in α-synuclein and inhibit fibrillation may be significant for engineering DNA-chip-based therapeutic approaches to PD and other amyloid disorders.
    28. 28
      Cherny, D.; Hoyer, W.; Subramaniam, V.; Jovin, T. M. Double-stranded DNA stimulates the fibrillation of alpha-synuclein in vitro and is associated with the mature fibrils: an electron microscopy study. J. Mol. Biol. 2004, 344 (4), 929– 938,  DOI: 10.1016/j.jmb.2004.09.096
      28
      Double-stranded DNA Stimulates the Fibrillation of α-Synuclein in vitro and is Associated with the Mature Fibrils: An Electron Microscopy Study
      Cherny, Dmitry; Hoyer, Wolfgang; Subramaniam, Vinod; Jovin, Thomas M.
      Journal of Molecular Biology (2004), 344 (4), 929-938CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
      Filamentous aggregates formed by α-synuclein are a prominent and presumably key etiol. factor in Parkinson's and other neurodegenerative diseases characterized by motor disorders. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation properties of α-synuclein, e.g. by accelerating the process of assembly. Histones, the major component and constituent of chromatin, interact specifically with α-synuclein and enhance its fibrillation significantly. Here, we report that another component of chromatin, double-stranded DNA (dsDNA), either linear or supercoiled, also interacts with wild-type α-synuclein, leading to a significant stimulation of α-synuclein assembly into mature fibrils characterized by a reduced lag phase. In general, the morphol. of the fibrils remains unchanged in the presence of linear dsDNA. Electron microscopy reveals that DNA forms various types of complexes upon assocn. with the fibrils at their surface without distortion of the double-helical structure. The existence of these complexes was confirmed by the electrophoresis, which also demonstrated that a fraction of the assocd. DNA was resistant to digestion by restriction endonucleases. Fibrils assembled from the α-synuclein mutants A30P and A53T and the C-terminally truncated variants (encoding amino acid residues 1-108 or 1-124) also form complexes with linear dsDNA. Possible mechanisms and implications of dsDNA-α-synuclein interactions are discussed.
    29. 29
      Jiang, K.; Rocha, S.; Westling, A.; Kesarimangalam, S.; Dorfman, K. D.; Wittung-Stafshede, P.; Westerlund, F. Alpha-Synuclein Modulates the Physical Properties of DNA. Chemistry 2018, 24 (58), 15685– 15690,  DOI: 10.1002/chem.201803933
      There is no corresponding record for this reference.
    30. 30
      Jiang, K.; Rocha, S.; Kumar, R.; Westerlund, F.; Wittung-Stafshede, P. C-terminal truncation of alpha-synuclein alters DNA structure from extension to compaction. Biochem. Biophys. Res. Commun. 2021, 568, 43– 47,  DOI: 10.1016/j.bbrc.2021.06.059
      There is no corresponding record for this reference.
    31. 31
      Weston, L. J.; Bowman, A. M.; Osterberg, V. R.; Meshul, C. K.; Woltjer, R. L.; Unni, V. K. Aggregated Alpha-Synuclein Inclusions within the Nucleus Predict Impending Neuronal Cell Death in a Mouse Model of Parkinsonism. Int. J. Mol. Sci. 2022, 23 (23), 15294,  DOI: 10.3390/ijms232315294
      There is no corresponding record for this reference.
    32. 32
      Koss, D. J.; Erskine, D.; Porter, A.; Palmoski, P.; Menon, H.; Todd, O. G. J.; Leite, M.; Attems, J.; Outeiro, T. F. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies. Acta Neuropathol. Commun. 2022, 10, 98,  DOI: 10.1186/s40478-022-01403-x
      There is no corresponding record for this reference.
    33. 33
      Nishie, M.; Mori, F.; Yoshimoto, M.; Takahashi, H.; Wakabayashi, K. A quantitative investigation of neuronal cytoplasmic and intranuclear inclusions in the pontine and inferior olivary nuclei in multiple system atrophy. Neuropathol. Appl. Neurobiol. 2004, 30 (5), 546– 554,  DOI: 10.1111/j.1365-2990.2004.00564.x
      There is no corresponding record for this reference.
    34. 34
      Lin, W.-L.; DeLucia, M. W.; Dickson, D. W. α-Synuclein immunoreactivity in neuronal nuclear inclusions and neurites in multiple system atrophy. Neurosci. Lett. 2004, 354 (2), 99– 102,  DOI: 10.1016/j.neulet.2003.09.075
      There is no corresponding record for this reference.
    35. 35
      Wittung-Stafshede, P. Chemical catalysis by biological amyloids. Biochem. Soc. Trans. 2023, 51 (5), 1967– 1974,  DOI: 10.1042/BST20230617
      There is no corresponding record for this reference.
    36. 36
      Horvath, I.; Wittung-Stafshede, P. Amyloid Fibers of α-Synuclein Catalyze Chemical Reactions. ACS Chem. Neurosci. 2023, 14, 603– 608,  DOI: 10.1021/acschemneuro.2c00799
      There is no corresponding record for this reference.
    37. 37
      Horvath, I.; Mohamed, K. A.; Kumar, R.; Wittung-Stafshede, P. Amyloids of alpha-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites. Int. J. Mol. Sci. 2023, 24 (16), 12849,  DOI: 10.3390/ijms241612849
      There is no corresponding record for this reference.
    38. 38
      Arad, E.; Baruch Leshem, A.; Rapaport, H.; Jelinek, R. β-Amyloid fibrils catalyze neurotransmitter degradation. Chem Catal. 2021, 1 (4), 908– 922,  DOI: 10.1016/j.checat.2021.07.005
      38
      β-Amyloid fibrils catalyze neurotransmitter degradation
      Arad, Elad; Baruch Leshem, Avigail; Rapaport, Hanna; Jelinek, Raz
      Chem Catalysis (2021), 1 (4), 908-922CODEN: CCHAE9; ISSN:2667-1093. (Elsevier Inc.)
      Amyloid fibrils are one of the hallmarks of Alzheimer's disease (AD), although a causative link between plaque-forming amyloid fibrils and AD pathol. remains to be clarified. This study demonstrates, for the first time for a naturally occurring amyloid, that fibrils comprising the 42-residue amyloid-β peptide (Aβ42) exhibit significant catalytic properties. Aβ42 fibrils catalyzed the hydrolysis of the model ester para-nitrophenyl acetate (pNPA) and of acetylthiocholine, a surrogate for the neurotransmitter acetylcholine. Aβ42 fibrils also catalyzed oxidn. of the prominent neurotransmitters dopamine and adrenaline. Importantly, the catalytic activity was specifically manifested by mature Aβ42 fibrils and not the peptide monomers or oligomeric Aβ42, the putative neurotoxic species. Furthermore, maximal catalytic activity was recorded by the full-length Aβ42 fibrils, whereas fibrillar assemblies comprising Aβ42 subdomains were significantly less catalytic. The catalytic activity of Aβ fibrils could exhibit insidious roles in AD pathophysiol.
    39. 39
      Arad, E.; Yosefi, G.; Kolusheva, S.; Bitton, R.; Rapaport, H.; Jelinek, R. Native Glucagon Amyloids Catalyze Key Metabolic Reactions. ACS Nano 2022, 16 (8), 12889– 12899,  DOI: 10.1021/acsnano.2c05166
      39
      Native Glucagon Amyloids Catalyze Key Metabolic Reactions
      Arad, Elad; Yosefi, Gal; Kolusheva, Sofiya; Bitton, Ronit; Rapaport, Hanna; Jelinek, Raz
      ACS Nano (2022), 16 (8), 12889-12899CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Glucagon is a prominent peptide hormone, playing central roles in the regulation of glucose blood-level and lipid metab. Formation of glucagon amyloid fibrils was previously reported, although no biol. functions of such fibrils are known. Here, glucagon amyloid fibrils catalyze biol. important reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. In particular, glucagon fibrils catalyze dephosphorylation of ATP, a core metabolic reaction in cell biol. Comparative anal. of several glucagon variants allowed mapping the catalytic activity to an enzymic pocket-like triad formed at the glucagon fibril surface, comprising the histidyl-serine domain at the N-terminus of the peptide. This study may point to previously unknown physiol. roles and pathol. consequences of glucagon fibrillation, and supports the hypothesis that catalytic activities of native amyloid fibrils play functional roles in human physiol. and disease.
    40. 40
      Theillet, F.-X.; Binolfi, A.; Bekei, B.; Martorana, A.; Rose, H. M.; Stuiver, M. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016, 530, 45,  DOI: 10.1038/nature16531
      40
      Structural disorder of monomeric α-synuclein persists in mammalian cells
      Theillet, Francois-Xavier; Binolfi, Andres; Bekei, Beata; Martorana, Andrea; Rose, Honor May; Stuiver, Marchel; Verzini, Silvia; Lorenz, Dorothea; van Rossum, Marleen; Goldfarb, Daniella; Selenko, Philipp
      Nature (London, United Kingdom) (2016), 530 (7588), 45-50CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Intracellular aggregation of the human amyloid protein, α-synuclein (I), is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here, the authors used NMR and ESR spectroscopy to derive at.-resoln. insights into the structure and dynamics of I in different mammalian cell types. The authors showed that the disordered nature of monomeric I was stably preserved in non-neuronal and neuronal cells. Under physiol. cell conditions, I was N-terminally acetylated and adopted conformations that were more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results established that different types of crowded intracellular environments do not inherently promote I oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.
    41. 41
      Cervantes, N. A. G.; Medina, B. G. Robust deposition of lambda DNA on mica for imaging by AFM in air. Scanning 2014, 36 (6), 561– 569,  DOI: 10.1002/sca.21155
      There is no corresponding record for this reference.
    42. 42
      Singh, V.; Johansson, P.; Ekedahl, E.; Lin, Y. L.; Hammarsten, O.; Westerlund, F. Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging. Biochem. Biophys. Res. Commun. 2022, 594, 57– 62,  DOI: 10.1016/j.bbrc.2022.01.041
      There is no corresponding record for this reference.
    43. 43
      Singh, V.; Johansson, P.; Lin, Y. L.; Hammarsten, O.; Westerlund, F. Shining light on single-strand lesions caused by the chemotherapy drug bleomycin. DNA Repair 2021, 105, 103153,  DOI: 10.1016/j.dnarep.2021.103153
      There is no corresponding record for this reference.
    44. 44
      Zirkin, S.; Fishman, S.; Sharim, H.; Michaeli, Y.; Don, J.; Ebenstein, Y. Lighting up individual DNA damage sites by in vitro repair synthesis. J. Am. Chem. Soc. 2014, 136 (21), 7771– 7776,  DOI: 10.1021/ja503677n
      44
      Lighting Up Individual DNA Damage Sites by In Vitro Repair Synthesis
      Zirkin, Shahar; Fishman, Sivan; Sharim, Hila; Michaeli, Yael; Don, Jeremy; Ebenstein, Yuval
      Journal of the American Chemical Society (2014), 136 (21), 7771-7776CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      DNA damage and repair are linked to fundamental biol. processes such as metab., disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-mol. imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quant. follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradn. in several cell types. Finally, we show the modularity of this single-mol. approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extd. from mouse brain tissue.
    45. 45
      Boiteux, S.; Guillet, M. Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae. DNA Repair 2004, 3 (1), 1– 12,  DOI: 10.1016/j.dnarep.2003.10.002
      45
      Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae
      Boiteux, Serge; Guillet, Marie
      DNA Repair (2004), 3 (1), 1-12CODEN: DRNEAR; ISSN:1568-7864. (Elsevier Science B.V.)
      A review. Apurinic/apyrimidinic (AP) sites are one of the most frequent spontaneous lesions in DNA. They are potentially mutagenic and lethal lesions that can block DNA replication and transcription. In addn., cleavage of AP sites by AP endonucleases or AP lyases generates DNA single-strand breaks (SSBs) with 5'- or 3'-blocked ends, resp. Therefore, we suggest that AP sites and 3'- or 5'-blocked SSBs, we name "honorary AP sites", constitute a single class of lesions. In this review, we describe the different mechanisms used by the budding yeast Saccharomyces cerevisiae to remove or tolerate AP sites and related SSBs. In wild-type cells, AP sites are primarily repaired by the base excision repair (BER) pathway, with the nucleotide excision repair (NER) pathway as a back up activity. BER is initiated by one of the two AP endonucleases, Apn1 or Apn2. Three DNA N-glycosylases/AP lyases, Ntg1, Ntg2 and Ogg1, can also incise AP sites in DNA. Rad27, a structure specific endonuclease, is involved in the repair of 5'-blocked ends, whereas Apn1, Apn2 and Rad1-Rad10 are involved in the removal of 3'-blocked ends using their 3'-phosphodiesterase and 3'-flap endonuclease activities, resp. AP sites can stall DNA replication forks, as well as they block in vitro DNA synthesis by DNA polymerase δ. Restart of stalled forks can occur through a recombination-assocd. pathway initiated by the Mus81-Mms4 endonuclease or mutagenic translesion DNA synthesis (TLS). The mutagenic bypass of AP sites is a two-polymerases affair with an inserter DNA polymerase (Polδ, Polη or Rev1) and an extender DNA polymerase (Polζ). Under normal growth conditions, inactivation of Apn1, Apn2 and Rad1-Rad10 causes cell death. Therefore, the burden of spontaneous AP sites is not compatible with life, in the absence of excision repair pathways. These results in yeast demonstrate that AP sites are crit. endogenous DNA damages that cause genetic instability and by analogy could be assocd. with degenerative pathologies in human.
    46. 46
      Nyberg, L.; Persson, F.; Akerman, B.; Westerlund, F. Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA. Nucleic Acids Res. 2013, 41 (19), e184  DOI: 10.1093/nar/gkt755
      There is no corresponding record for this reference.
    47. 47
      Ischenko, A. A.; Saparbaev, M. K. Alternative nucleotide incision repair pathway for oxidative DNA damage. Nature 2002, 415 (6868), 183– 187,  DOI: 10.1038/415183a
      There is no corresponding record for this reference.
    48. 48
      Yang, J. L.; Chen, W. Y.; Mukda, S.; Yang, Y. R.; Sun, S. F.; Chen, S. D. Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons. Neuropathol. Appl. Neurobiol. 2020, 46 (4), 375– 390,  DOI: 10.1111/nan.12584
      There is no corresponding record for this reference.
    49. 49
      Thakur, S.; Sarkar, B.; Cholia, R. P.; Gautam, N.; Dhiman, M.; Mantha, A. K. APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions. Exp. Mol. Med. 2014, 46 (7), e106  DOI: 10.1038/emm.2014.42
      49
      APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions
      Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P.; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K.
      Experimental & Molecular Medicine (2014), 46 (7), e106CODEN: EMMEF3; ISSN:2092-6413. (NPG Nature Asia-Pacific)
      A review. Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiol. processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metab., cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addn., studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.
    50. 50
      Almeida, K. H.; Sobol, R. W. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. DNA Repair 2007, 6 (6), 695– 711,  DOI: 10.1016/j.dnarep.2007.01.009
      There is no corresponding record for this reference.
    51. 51
      Hegde, M. L.; Izumi, T.; Mitra, S. Oxidized base damage and single-strand break repair in mammalian genomes: role of disordered regions and posttranslational modifications in early enzymes. Prog. Mol. Biol. Transl. Sci. 2012, 110, 123– 153,  DOI: 10.1016/B978-0-12-387665-2.00006-7
      There is no corresponding record for this reference.
    52. 52
      Oz, R.; Kk, S.; Westerlund, F. A nanofluidic device for real-time visualization of DNA-protein interactions on the single DNA molecule level. Nanoscale 2019, 11 (4), 2071– 2078,  DOI: 10.1039/C8NR09023H
      There is no corresponding record for this reference.
    53. 53
      Estévez-Torres, A.; Baigl, D. DNA compaction: fundamentals and applications. Soft Matter 2011, 7 (15), 6746– 6756,  DOI: 10.1039/c1sm05373f
      53
      DNA compaction: fundamentals and applications
      Estevez-Torres, Andre; Baigl, Damien
      Soft Matter (2011), 7 (15), 6746-6756CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      A review. Compaction is the process in which a large DNA mol. undergoes a transition between an elongated conformation and a very compact form. In nature, DNA compaction occurs to package genomic material inside tiny spaces such as viral capsids and cell nuclei. In vitro, several strategies exist to compact DNA. In this review, we first provide a physico-chem. description of this phenomenon, focusing on the modes of compaction, the types of compaction agents and the chem. and phys. parameters that control compaction and its reverse process, decompaction. We then describe three main kinds of applications. First, we show how regulated compaction/decompaction can be used to control gene activity in vitro, with a particular emphasis on the use of light to reversibly control gene expression. Second, we describe several approaches where compaction is used as a way to reversibly protect DNA against chem., biochem., or mech. stresses. Third, we show that compact DNA can be used as a nanostructure template to generate nanomaterials with a well-defined size and shape. We conclude by proposing some perspectives for future biochem. and biotechnol. applications and enumerate some remaining challenges that we think worth being undertaken.
    54. 54
      van der Maarel, J. R. C.; Zhang, C.; van Kan, J. A. A Nanochannel Platform for Single DNA Studies: From Crowding, Protein DNA Interaction, to Sequencing of Genomic Information. Isr. J. Chem. 2014, 54 (11–12), 1573– 1588,  DOI: 10.1002/ijch.201400091
      There is no corresponding record for this reference.
    55. 55
      Jiang, K.; Humbert, N.; Kk, S.; Rouzina, I.; Mely, Y.; Westerlund, F. The HIV-1 nucleocapsid chaperone protein forms locally compacted globules on long double-stranded DNA. Nucleic Acids Res. 2021, 49 (8), 4550– 4563,  DOI: 10.1093/nar/gkab236
      There is no corresponding record for this reference.
    56. 56
      Sharma, R.; Kk, S.; Holmstrom, E. D.; Westerlund, F. Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion. Biochem. Biophys. Res. Commun. 2020, 533 (1), 175– 180,  DOI: 10.1016/j.bbrc.2020.06.051
      There is no corresponding record for this reference.
    57. 57
      Wood, S. J.; Wypych, J.; Steavenson, S.; Louis, J. C.; Citron, M.; Biere, A. L. alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease. J. Biol. Chem. 1999, 274 (28), 19509– 19512,  DOI: 10.1074/jbc.274.28.19509
      57
      α-Synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease
      Wood, Stephen J.; Wypych, Jette; Steavenson, Shirley; Louis, Jean-Claude; Citron, Martin; Biere, Anja Leona
      Journal of Biological Chemistry (1999), 274 (28), 19509-19512CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Parkinson's disease (PD) is a neurodegenerative disorder that is pathol. characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD. α-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and the authors have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of α-synuclein aggregation: the results show that (i) it is a nucleation-dependent process that can be seeded by aggregated α-synuclein functioning as nuclei; (ii) this fibril growth follows first-order kinetics with respect to α-synuclein concn.; (iii) mutant α-synuclein can seed the aggregation of wild-type α-synuclein, which leads to the prediction that the Lewy bodies of familial PD patients with α-synuclein mutations will contain both the mutant and the wild-type protein; and (iv) wild-type and mutant forms of α-synuclein do not differ in their crit. concns. Apparently, differences in aggregation kinetics of α-synucleins cannot be explained by differences in soly. but are due to different nucleation rates. Consequently, α-synuclein nucleation may be the rate-limiting step for the formation of Lewy body α-synuclein fibrils in Parkinson's disease.
    58. 58
      Tkach, J. M.; Yimit, A.; Lee, A. Y.; Riffle, M.; Costanzo, M.; Jaschob, D. Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress. Nat. Cell Biol. 2012, 14 (9), 966– 976,  DOI: 10.1038/ncb2549
      58
      Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress
      Tkach, Johnny M.; Yimit, Askar; Lee, Anna Y.; Riffle, Michael; Costanzo, Michael; Jaschob, Daniel; Hendry, Jason A.; Ou, Jiongwen; Moffat, Jason; Boone, Charles; Davis, Trisha N.; Nislow, Corey; Brown, Grant W.
      Nature Cell Biology (2012), 14 (9), 966-976CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chem. or genetic stress to reveal cellular response pathways.
    59. 59
      Franssens, V.; Boelen, E.; Anandhakumar, J.; Vanhelmont, T.; Büttner, S.; Winderickx, J. Yeast unfolds the road map toward α-synuclein-induced cell death. Cell Death Differ. 2010, 17 (5), 746– 753,  DOI: 10.1038/cdd.2009.203
      There is no corresponding record for this reference.
    60. 60
      Jenkins, W. T.; Marshall, M. M. A modified direct phosphate assay for studying ATPases. Anal. Biochem. 1984, 141 (1), 155– 160,  DOI: 10.1016/0003-2697(84)90439-1
      There is no corresponding record for this reference.
    61. 61
      Zhang, S.; Li, J.; Xu, Q.; Xia, W.; Tao, Y.; Shi, C. Conformational Dynamics of an alpha-Synuclein Fibril upon Receptor Binding Revealed by Insensitive Nuclei Enhanced by Polarization Transfer-Based Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy. J. Am. Chem. Soc. 2023, 145 (8), 4473– 4484,  DOI: 10.1021/jacs.2c10854
      There is no corresponding record for this reference.
    62. 62
      Gezen-Ak, D.; Yurttas, Z.; Camoglu, T.; Dursun, E. Could Amyloid-beta 1–42 or alpha-Synuclein Interact Directly with Mitochondrial DNA? A Hypothesis. ACS Chem. Neurosci. 2022, 13 (19), 2803– 2812,  DOI: 10.1021/acschemneuro.2c00512
      There is no corresponding record for this reference.
    63. 63
      Werner, T.; Kumar, R.; Horvath, I.; Scheers, N.; Wittung-Stafshede, P. Abundant fish protein inhibits α-synuclein amyloid formation. Sci. Rep. 2018, 8, 5465,  DOI: 10.1038/s41598-018-23850-0
      There is no corresponding record for this reference.
    64. 64
      Oz, R.; Wang, J. L.; Guerois, R.; Goyal, G.; Kk, S.; Ropars, V. Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis. Nucleic Acids Res. 2021, 49 (5), 2629– 2641,  DOI: 10.1093/nar/gkab083
      There is no corresponding record for this reference.
    65. 65
      Heenan, P. R.; Perkins, T. T. Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions. ACS Nano 2019, 13 (4), 4220– 4229,  DOI: 10.1021/acsnano.8b09234
      65
      Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions
      Heenan, Patrick R.; Perkins, Thomas T.
      ACS Nano (2019), 13 (4), 4220-4229CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      For over 25 years, imaging of DNA by at. force microscopy has been intensely pursued. Ideally, such images are then used to probe the phys. properties of DNA and characterize protein-DNA interactions. The at. flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the neg. charge of mica and DNA hinders deposition. Traditional methods for imaging DNA and protein-DNA complexes in liq. have drawbacks: DNA conformations with an anomalous persistence length (p), low SNR, and/or ionic deposition conditions detrimental to preserving protein-DNA interactions. Here, we developed a process to bind DNA to mica in a buffer contg. both MgCl2 and KCl that resulted in high SNR images of equilibrated DNA in liq. Achieving an equilibrated 2D configuration (i.e., p = 50 nm) not only implied a minimally perturbative binding process but also improved data quality and quantity because the DNA's configuration was more extended. In comparison to a purely NiCl2-based protocol, we showed that an 8-fold larger fraction (90%) of 680-nm-long DNA mols. could be quantified. High-resoln. images of select equilibrated mols. revealed the right-handed structure of DNA with a helical pitch of 3.5 nm. Deposition and imaging of DNA was achieved over a wide range of monovalent and divalent ionic conditions, including a buffer contg. 50 mM KCl and 3 mM MgCl2. Finally, we imaged two protein-DNA complexes using this protocol: a restriction enzyme bound to DNA and a small three-nucleosome array. We expect such deposition of protein-DNA complexes at biochem. relevant ionic conditions will facilitate biophys. insights derived from imaging diverse protein-DNA complexes.
    66. 66
      Horcas, I.; Fernández, R.; Gómez-Rodríguez, J. M.; Colchero, J.; Gómez-Herrero, J.; Baro, A. M. WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 2007, 78, 013705,  DOI: 10.1063/1.2432410
      66
      WSXM: a software for scanning probe microscopy and a tool for nanotechnology
      Horcas, I.; Fernandez, R.; Gomez-Rodriguez, J. M.; Colchero, J.; Gomez-Herrero, J.; Baro, A. M.
      Review of Scientific Instruments (2007), 78 (1), 013705/1-013705/8CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
      In this work we briefly describe the most relevant features of WSXM, a freeware scanning probe microscopy software based on MS-Windows. The article is structured in three different sections: The introduction is a perspective on the importance of software on scanning probe microscopy. The second section is devoted to describe the general structure of the application; in this section the capabilities of WSXM to read third party files are stressed. Finally, a detailed discussion of some relevant procedures of the software is carried out.
    67. 67
      Kk, S.; Persson, F.; Fritzsche, J.; Beech, J. P.; Tegenfeldt, J. O.; Westerlund, F. Fluorescence Microscopy of Nanochannel-Confined DNA. In Single Molecule Analysis: Methods and Protocols; Heller, I., Dulin, D., Peterman, E. J. G., Eds.; Springer US: New York, NY, 2024; pp 175– 202.
      There is no corresponding record for this reference.
    68. 68
      Frykholm, K.; Müller, V.; Kk, S.; Dorfman, K. D.; Westerlund, F. DNA in nanochannels: theory and applications. Q. Rev. Biophys. 2022, 55, e12  DOI: 10.1017/S0033583522000117
      There is no corresponding record for this reference.
    69. 69
      Sampaio-Marques, B.; Felgueiras, C.; Silva, A.; Rodrigues, M.; Tenreiro, S.; Franssens, V. SNCA (α-synuclein)-induced toxicity in yeast cells is dependent on Sir2-mediated mitophagy. Autophagy 2012, 8 (10), 1494– 1509,  DOI: 10.4161/auto.21275
      There is no corresponding record for this reference.
    70. 70
      Hanzén, S.; Vielfort, K.; Yang, J.; Roger, F.; Andersson, V.; Zamarbide-Forés, S. Lifespan Control by Redox-Dependent Recruitment of Chaperones to Misfolded Proteins. Cell 2016, 166 (1), 140– 151,  DOI: 10.1016/j.cell.2016.05.006
      There is no corresponding record for this reference.
    71. 71
      Andersson, R.; Eisele-Bürger, A. M.; Hanzén, S.; Vielfort, K.; Öling, D.; Eisele, F. Differential role of cytosolic Hsp70s in longevity assurance and protein quality control. PLoS Genet. 2021, 17 (1), e1008951  DOI: 10.1371/journal.pgen.1008951
      71
      Differential role of cytosolic Hsp70s in longevity assurance and protein quality control
      Andersson, Rebecca; Eisele-Burger, Anna Maria; Hanzen, Sarah; Vielfort, Katarina; Oeling, David; Eisele, Frederik; Johansson, Gustav; Gustafsson, Tobias; Kvint, Kristian; Nystroem, Thomas
      PLoS Genetics (2021), 17 (1), e1008951CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)
      70 KDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe redn. in cellular reprodn. and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degrdn. In contrast, Ssa4 overprodn. in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-assocd. aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overprodn. extended the short lifespan of ssa1Δ ssa2Δ mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast.
    72. 72
      Outeiro, T. F.; Lindquist, S. Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology. Science 2003, 302 (5651), 1772– 1775,  DOI: 10.1126/science.1090439
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.4c00461.

    • Figure S1 (SPR sensograms for binding), Figure S2 (additional AFM images of αS and DNA), Figure S3 [DNA damage induced by αS(1–119) amyloids], Figure S4 (DNA length distributions from analysis of DNA on coverslips), Figure S5 (ATPase activity for αS amyloids), and Figure S6 (high-resolution structures of the αS amyloid fold with putative DNA-binding site indicated) (PDF)


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