Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties
Abstract: Alpha-synuclein (aSyn) is a vertebrate protein, normally found within the presynaptic nerve terminal and nucleus, which is known to form somatic and neuritic aggregates in certain neurodegenerative diseases. Disease-associated aggregates of aSyn are heavily phosphorylated at serine-129 (pSyn), while normal aSyn protein is not. Within the nucleus, aSyn can directly bind DNA, but the mechanism of binding and the potential modulatory roles of phosphorylation are poorly understood. Here we demonstrate using a combination of electrophoretic mobility shift assay and atomic force microscopy approaches that both aSyn and pSyn can bind DNA within the major groove, in a DNA length-dependent manner and with little specificity for DNA sequence. Our data are consistent with a model in which multiple aSyn molecules bind a single 300 base pair (bp) DNA molecule in such a way that stabilizes the DNA in a bent conformation. We propose that serine-129 phosphorylation decreases the ability of aSyn to both bind and bend DNA, as aSyn binds 304 bp circular DNA forced into a bent shape, but pSyn does not. Two aSyn paralogs, beta- and gamma-synuclein, also interact with DNA differently than aSyn, and do not stabilize similar DNA conformations. Our work suggests that reductions in aSyn's ability to bind and bend DNA induced by serine-129 phosphorylation may be important for modulating aSyn's known roles in DNA metabolism, including the regulation of transcription and DNA repair. Alpha-synuclein (aSyn) is a vertebrate protein, normally found within the presynaptic nerve terminal and nucleus, which is known to form somatic and neuritic aggregates in certain neurodegenerative diseases. Disease-associated aggregates of aSyn are heavily phosphorylated at serine-129 (pSyn), while normal aSyn protein is not. Within the nucleus, aSyn can directly bind DNA, but the mechanism of binding and the potential modulatory roles of phosphorylation are poorly understood. Here we demonstrate using a combination of electrophoretic mobility shift assay and atomic force microscopy approaches that both aSyn and pSyn can bind DNA within the major groove, in a DNA length-dependent manner and with little specificity for DNA sequence. Our data are consistent with a model in which multiple aSyn molecules bind a single 300 base pair (bp) DNA molecule in such a way that stabilizes the DNA in a bent conformation. We propose that serine-129 phosphorylation decreases the ability of aSyn to both bind and bend DNA, as aSyn binds 304 bp circular DNA forced into a bent shape, but pSyn does not. Two aSyn paralogs, beta- and gamma-synuclein, also interact with DNA differently than aSyn, and do not stabilize similar DNA conformations. Our work suggests that reductions in aSyn's ability to bind and bend DNA induced by serine-129 phosphorylation may be important for modulating aSyn's known roles in DNA metabolism, including the regulation of transcription and DNA repair. Alpha-synuclein (aSyn) is a 140 amino acid–long protein found in vertebrates, where it is known to localize to the presynaptic nerve terminal (1Maroteaux L. Campanelli J.T. Scheller R.H. Synuclein: A neuron-specific protein localized to the nucleus and presynaptic nerve terminal.J. Neurosci. 1988; 8: 2804-2815Google Scholar, 2Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. de Silva H.A. Kittel A. Saitoh T. The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system.Neuron. 1995; 14: 467-475Google Scholar, 3George J.M. Jin H. Woods W.S. Clayton D.F. Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.Neuron. 1995; 15: 361-372Google Scholar) and nucleus (1Maroteaux L. Campanelli J.T. Scheller R.H. Synuclein: A neuron-specific protein localized to the nucleus and presynaptic nerve terminal.J. Neurosci. 1988; 8: 2804-2815Google Scholar, 4Goers J. Manning-Bog A.B. McCormack A.L. Millett I.S. Doniach S. Di Monte D.A. Uversky V.N. Fink A.L. Nuclear localization of alpha-synuclein and its interaction with histones.Biochemistry. 2003; 42: 8465-8471Google Scholar, 5Yu S. Li X. Liu G. Han J. Zhang C. Li Y. Xu S. Liu C. Gao Y. Yang H. Uéda K. Chan P. Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody.Neuroscience. 2007; 145: 539-555Google Scholar, 6Schell H. Hasegawa T. Neumann M. Kahle P.J. Nuclear and neuritic distribution of serine-129 phosphorylated alpha-synuclein in transgenic mice.Neuroscience. 2009; 160: 796-804Google Scholar, 7Pinho R. Paiva I. Jercic K.G. Fonseca-Ornelas L. Gerhardt E. Fahlbusch C. Garcia-Esparcia P. Kerimoglu C. Pavlou M.A. Villar-Pique A. Szego É. Lopes da Fonseca T. Odoardi F. Soeroes S. Rego A.C. et al.Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.Hum. Mol. Genet. 2019; 28: 31-50Google Scholar, 8Ma K.L. Song L.K. Yuan Y.H. Zhang Y. Han N. Gao K. Chen N.H. The nuclear accumulation of alpha-synuclein is mediated by importin alpha and promotes neurotoxicity by accelerating the cell cycle.Neuropharmacology. 2014; 82: 132-142Google Scholar, 9Schaser 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. Raber J. Luk K.C. McCullough A.K. Woltjer R.L. Unni V.K. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.Sci. Rep. 2019; 9: 10919Google Scholar). aSyn is linked to genetic forms of parkinsonism in families with an autosomal dominant inheritance pattern. The first such point mutation found (A53T) was in the N-terminal phospholipid-binding domain (10Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. et al.Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.Science. 1997; 276: 2045-2047Google Scholar). Since then, five additional point mutations (A30P, E46K, H50Q, G51D, A53E) have been suggested to cause parkinsonism (11Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease.Nat. Genet. 1998; 18: 106-108Google Scholar, 12Zarranz J.J. Alegre J. Gomez-Esteban J.C. Lezcano E. Ros R. Ampuero I. Vidal L. Hoenicka J. Rodriguez O. Atares B. Llorens V. Gomez Tortosa E. del Ser T. Muñoz D.G. de Yebenes J.G. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia.Ann. Neurol. 2004; 55: 164-173Google Scholar, 13Appel-Cresswell S. Vilarino-Guell C. Encarnacion M. Sherman H. Yu I. Shah B. Weir D. Thompson C. Szu-Tu C. Trinh J. Aasly J.O. Rajput A. Rajput A.H. Jon Stoessl A. Farrer M.J. Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease.Mov. Disord. 2013; 28: 811-813Google Scholar, 14Proukakis C. Dudzik C.G. Brier T. MacKay D.S. Cooper J.M. Millhauser G.L. Houlden H. Schapira A.H. A novel α-synuclein missense mutation in Parkinson disease.Neurology. 2013; 80: 1062-1064Google Scholar, 15Lesage S. Anheim M. Letournel F. Bousset L. Honore A. Rozas N. Pieri L. Madiona K. Durr A. Melki R. Verny C. Brice A. G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome.Ann. Neurol. 2013; 73: 459-471Google Scholar, 16Kiely A.P. Asi Y.T. Kara E. Limousin P. Ling H. Lewis P. Proukakis C. Quinn N. Lees A.J. Hardy J. Revesz T. Houlden H. Holton J.L. α-Synucleinopathy associated with G51D SNCA mutation: A link between Parkinson's disease and multiple system atrophy?.Acta Neuropathol. 2013; 125: 753-769Google Scholar, 17Pasanen P. Myllykangas L. Siitonen M. Raunio A. Kaakkola S. Lyytinen J. Tienari P.J. Poyhonen M. Paetau A. Novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology.Neurobiol. Aging. 2014; 35: 2180.e1-2180.e5Google Scholar), and all occur within a 24 amino acid stretch of the N-terminal phospholipid-binding domain, indicating the potential importance of this region in triggering disease. This initial genetic linkage between aSyn and parkinsonism led to the discovery that the pathological hallmark of sporadic (nongenetic) Parkinson's Disease, the Lewy body, is composed primarily of misfolded aSyn (18Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Alpha-synuclein in Lewy bodies.Nature. 1997; 388: 839-840Google Scholar), in a combination of fibrillar and granular aggregation states (19Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies.Am. J. Pathol. 1998; 152: 879-884Google Scholar, 20Forno L.S. Neuropathology of Parkinson's disease.J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Google Scholar). A subset of prominent neurodegenerative conditions in addition to Parkinson's Disease, including Dementia with Lewy bodies, Multiple System Atrophy, and Pure Autonomic Failure, are typified by the formation of aSyn aggregates. This has led to the adoption of the term "synucleinopathy" to describe the group of diseases characterized by pathological aggregation of aSyn. Interestingly, increased expression of aSyn has been shown to occur in certain cancers, most prominently in the skin cancer melanoma (21Matsuo Y. Kamitani T. Parkinson's disease-related protein, alpha-synuclein, in malignant melanoma.PLoS One. 2010; 5e10481Google Scholar, 22Lee B.R. Matsuo Y. Cashikar A.G. Kamitani T. Role of Ser129 phosphorylation of α-synuclein in melanoma cells.J. Cell Sci. 2013; 126: 696-704Google Scholar, 23Welinder C. Jonsson G.B. Ingvar C. Lundgren L. Baldetorp B. Olsson H. Breslin T. Rezeli M. Jansson B. Fehniger T.E. Laurell T. Wieslander E. Pawlowski K. Marko-Varga G. Analysis of alpha-synuclein in malignant melanoma - development of a SRM quantification assay.PLoS One. 2014; 9e110804Google Scholar), a cancer with increased incidence in Parkinson's Disease patients (24Olsen J.H. Friis S. Frederiksen K. Malignant melanoma and other types of cancer preceding Parkinson disease.Epidemiology. 2006; 17: 582-587Google Scholar, 25Bajaj A. Driver J.A. Schernhammer E.S. Parkinson's disease and cancer risk: A systematic review and meta-analysis.Cancer Causes Control. 2010; 21: 697-707Google Scholar, 26Rugbjerg K. Friis S. Lassen C.F. Ritz B. Olsen J.H. Malignant melanoma, breast cancer and other cancers in patients with Parkinson's disease.Int. J. Cancer. 2012; 131: 1904-1911Google Scholar, 27Kareus S.A. Figueroa K.P. Cannon-Albright L.A. Pulst S.M. Shared predispositions of parkinsonism and cancer: A population-based pedigree-linked study.Arch. Neurol. 2012; 69: 1572-1577Google Scholar, 28Constantinescu R. Elm J. Auinger P. Sharma S. Augustine E.F. Khadim L. Kieburtz K. Investigators N.-P. Malignant melanoma in early-treated Parkinson's disease: The NET-PD trial.Mov. Disord. 2014; 29: 263-265Google Scholar, 29Ye Q. Wen Y. Al-Kuwari N. Chen X. Association between Parkinson's disease and melanoma: Putting the pieces together.Front. Aging Neurosci. 2020; 12: 60Google Scholar). aSyn's roles within the presynaptic terminal have been extensively explored and multiple specific functions in regulating chemical neurotransmission within this subcellular compartment have been described, including modulating exocytosis (30Larsen K.E. Schmitz Y. Troyer M.D. Mosharov E. Dietrich P. Quazi A.Z. Savalle M. Nemani V. Chaudhry F.A. Edwards R.H. Stefanis L. Sulzer D. Alpha-synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis.J. Neurosci. 2006; 26: 11915-11922Google Scholar, 31Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.Neuron. 2000; 25: 239-252Google Scholar), endocytosis (32Vargas K.J. Makani S. Davis T. Westphal C.H. Castillo P.E. Chandra S.S. Synucleins regulate the kinetics of synaptic vesicle endocytosis.J. Neurosci. 2014; 34: 9364-9376Google Scholar, 33Schechter M. Atias M. Abd Elhadi S. Davidi D. Gitler D. Sharon R. α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate.J. Biol. Chem. 2020; 295: 18076-18090Google Scholar), SNARE complex assembly (34Burre J. Sharma M. Tsetsenis T. Buchman V. Etherton M.R. Sudhof T.C. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro.Science. 2010; 329: 1663-1667Google Scholar), and vesicle clustering (35Nemani V.M. Lu W. Berge V. Nakamura K. Onoa B. Lee M.K. Chaudhry F.A. Nicoll R.A. Edwards R.H. Increased expression of alpha-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis.Neuron. 2010; 65: 66-79Google Scholar, 36Diao J. Burre J. Vivona S. Cipriano D.J. Sharma M. Kyoung M. Sudhof T.C. Brunger A.T. Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2.Elife. 2013; 2e00592Google Scholar, 37Wang L. Das U. Scott D.A. Tang Y. McLean P.J. Roy S. α-Synuclein multimers cluster synaptic vesicles and attenuate recycling.Curr. Biol. 2014; 24: 2319-2326Google Scholar). Several studies have shown that aSyn prefers binding highly curved, negatively charged phospholipid membranes through its N-terminal domain that associates with the outer leaflet of the convex-curved phospholipid bilayer of synaptic vesicles by adopting a complementary, concave-curved alpha-helical structure composed of two antiparallel helices (38Davidson W.S. Jonas A. Clayton D.F. George J.M. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.J. Biol. Chem. 1998; 273: 9443-9449Google Scholar, 39Chandra S. Chen X. Rizo J. Jahn R. Sudhof T.C. A broken alpha-helix in folded alpha-synuclein.J. Biol. Chem. 2003; 278: 15313-15318Google Scholar, 40Ulmer T.S. Bax A. Cole N.B. Nussbaum R.L. Structure and dynamics of micelle-bound human alpha-synuclein.J. Biol. Chem. 2005; 280: 9595-9603Google Scholar). It is through these interactions with neurotransmitter vesicles that aSyn is thought to mediate its presynaptic functions. In addition to its functions in the presynaptic terminal, aSyn can also localize to the nucleus where it is less well studied but has been suggested to modulate several DNA- and RNA-dependent processes. These include regulation of histone modification (4Goers J. Manning-Bog A.B. McCormack A.L. Millett I.S. Doniach S. Di Monte D.A. Uversky V.N. Fink A.L. Nuclear localization of alpha-synuclein and its interaction with histones.Biochemistry. 2003; 42: 8465-8471Google Scholar, 41Kontopoulos 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: 3012-3023Google Scholar), transcription (42Siddiqui A. Chinta S.J. Mallajosyula J.K. Rajagopolan S. Hanson I. Rane A. Melov S. Andersen J.K. 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 Radic. Biol. Med. 2012; 53: 993-1003Google Scholar, 43Kim S. Park J.M. Moon J. Choi H.J. Alpha-synuclein interferes with cAMP/PKA-dependent upregulation of dopamine β-hydroxylase and is associated with abnormal adaptive responses to immobilization stress.Exp. Neurol. 2014; 252: 63-74Google Scholar), rRNA (44Popova B. Wang D. Patz C. Akkermann D. Lazaro D.F. Galka D. Kolog Gulko M. Bohnsack M.T. Mobius W. Bohnsack K.E. Outeiro T.F. Braus G.H. DEAD-box RNA helicase Dbp4/DDX10 is an enhancer of α-synuclein toxicity and oligomerization.PLoS Genet. 2021; 17e1009407Google Scholar) and mRNA (45Chung C.Y. Khurana V. Yi S. Sahni N. Loh K.H. Auluck P.K. Baru V. Udeshi N.D. Freyzon Y. Carr S.A. Hill D.E. Vidal M. Ting A.Y. Lindquist S. In situ peroxidase labeling and mass-spectrometry connects alpha-synuclein directly to endocytic trafficking and mRNA metabolism in neurons.Cell Syst. 2017; 4: 242-250.e4Google Scholar, 46Khurana V. Peng J. Chung C.Y. Auluck P.K. Fanning S. Tardiff D.F. Bartels T. Koeva M. Eichhorn S.W. Benyamini H. Lou Y. Nutter-Upham A. Baru V. Freyzon Y. Tuncbag N. et al.Genome-scale networks link neurodegenerative disease genes to α-synuclein through specific molecular pathways.Cell Syst. 2017; 4: 157-170.e14Google Scholar) metabolism. Our recent work also suggests a previously unrecognized role for aSyn in modulating DNA double-strand break (DSB) repair (9Schaser 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. Raber J. Luk K.C. McCullough A.K. Woltjer R.L. Unni V.K. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.Sci. Rep. 2019; 9: 10919Google Scholar). Many of these potential functions are dependent on aSyn's ability to bind double-stranded nucleic acids, including DNA. Early work demonstrated that aSyn can bind DNA in its supercoiled form (47Hegde M.L. Jagannatha Rao K.S. Challenges and complexities of alpha-synuclein toxicity: New postulates in unfolding the mystery associated with Parkinson's disease.Arch. Biochem. Biophys. 2003; 418: 169-178Google Scholar) and that this binding can induce conformational changes both in the protein, increasing alpha-helical content (48Hegde M.L. Rao K.S. DNA induces folding in alpha-synuclein: Understanding the mechanism using chaperone property of osmolytes.Arch. Biochem. Biophys. 2007; 464: 57-69Google Scholar), and in bound DNA, converting it into an altered B form (48Hegde M.L. Rao K.S. DNA induces folding in alpha-synuclein: Understanding the mechanism using chaperone property of osmolytes.Arch. Biochem. Biophys. 2007; 464: 57-69Google Scholar, 49Vasudevaraju P. Guerrero E. Hegde M.L. Collen T.B. Britton G.B. Rao K.S. New evidence on α-synuclein and Tau binding to conformation and sequence specific GC∗ rich DNA: Relevance to neurological disorders.J. Pharm. Bioallied Sci. 2012; 4: 112-117Google Scholar). aSyn can also directly bind a large subset of DNA promoter sequences (7Pinho R. Paiva I. Jercic K.G. Fonseca-Ornelas L. Gerhardt E. Fahlbusch C. Garcia-Esparcia P. Kerimoglu C. Pavlou M.A. Villar-Pique A. Szego É. Lopes da Fonseca T. Odoardi F. Soeroes S. Rego A.C. et al.Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.Hum. Mol. Genet. 2019; 28: 31-50Google Scholar), including the PGC-1a (42Siddiqui A. Chinta S.J. Mallajosyula J.K. Rajagopolan S. Hanson I. Rane A. Melov S. Andersen J.K. 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 Radic. Biol. Med. 2012; 53: 993-1003Google Scholar) and Notch (50Desplats P. Spencer B. Crews L. Pathel P. Morvinski-Friedmann D. Kosberg K. Roberts S. Patrick C. Winner B. Winkler J. Masliah E. α-Synuclein induces alterations in adult neurogenesis in Parkinson disease models via p53-mediated repression of Notch1.J. Biol. Chem. 2012; 287: 31691-31702Google Scholar) gene promoters, where it appears to often downregulate transcription. NMR spectroscopy demonstrates that the same N-terminal domain that binds curved, negatively charged phospholipid membranes mediates aSyn's binding to the phosphate backbone of DNA (7Pinho R. Paiva I. Jercic K.G. Fonseca-Ornelas L. Gerhardt E. Fahlbusch C. Garcia-Esparcia P. Kerimoglu C. Pavlou M.A. Villar-Pique A. Szego É. Lopes da Fonseca T. Odoardi F. Soeroes S. Rego A.C. et al.Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.Hum. Mol. Genet. 2019; 28: 31-50Google Scholar). Single-molecule techniques have shown that aSyn binding can stretch DNA and increase its stiffness (51Jiang 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: 15685-15690Google Scholar, 52Jiang K. Rocha S. Kumar R. Westerlund F. Wittung-Stafshede P. C-terminal truncation of α-synuclein alters DNA structure from extension to compaction.Biochem. Biophys. Res. Commun. 2021; 568: 43-47Google Scholar), while fluorescence resonance energy transfer (FRET) assays developed to study noncanonical DNA structures show that aSyn stabilizes intermediate states on the pathway to DNA hairpin formation (53Mukherjee S.K. Knop J.M. Mobitz S. Winter R.H.A. Alteration of the conformational dynamics of a DNA hairpin by α-synuclein in the presence of aqueous two-phase systems.Chemistry. 2020; 26: 10987-10991Google Scholar) and can bind to i-motifs (54Mukherjee S.K. Knop J.M. Oliva R. Mobitz S. Winter R. Untangling the interaction of α-synuclein with DNA i-motifs and hairpins by volume-sensitive single-molecule FRET spectroscopy.RSC Chem. Biol. 2021; 2: 1196-1200Google Scholar). Although this work has started to reveal the mechanism of aSyn binding to DNA, important questions remain, including the role of potential disease-relevant aSyn posttranslational modifications in this process. Phosphorylation of aSyn at serine-129 (pSyn) is a well-established marker of pathological forms of aSyn aggregates. While <4% of aSyn is phosphorylated at this site in normal brain, within Lewy bodies >90% of aSyn bears this posttranslational modification (55Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. et al.Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.J. Biol. Chem. 2006; 281: 29739-29752Google Scholar). In addition, human autopsy studies have shown that pSyn is the most sensitive immunohistochemical marker yet developed to detect Lewy body pathology (56Beach T.G. White C.L. Hamilton R.L. Duda J.E. Iwatsubo T. Dickson D.W. Leverenz J.B. Roncaroli F. Buttini M. Hladik C.L. Sue L.I. Noorigian J.V. Adler C.H. Evaluation of alpha-synuclein immunohistochemical methods used by invited experts.Acta Neuropathol. 2008; 116: 277-288Google Scholar). Although pSyn may be important for disease pathogenesis, and previous work suggests that aSyn phosphorylation can modulate its nuclear localization (7Pinho R. Paiva I. Jercic K.G. Fonseca-Ornelas L. Gerhardt E. Fahlbusch C. Garcia-Esparcia P. Kerimoglu C. Pavlou M.A. Villar-Pique A. Szego É. Lopes da Fonseca T. Odoardi F. Soeroes S. Rego A.C. et al.Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.Hum. Mol. Genet. 2019; 28: 31-50Google Scholar, 57Goncalves S. Outeiro T.F. Assessing the subcellular dynamics of alpha-synuclein using photoactivation microscopy.Mol. Neurobiol. 2013; 47: 1081-1092Google Scholar, 58Weston L.J. Cook Z.T. Stackhouse T.L. Sal M.K. Schultz B.I. Tobias Z.J.C. Osterberg V.R. Brockway N.L. Pizano S. Glover G. Weissman T.A. Unni V.K. In vivo aggregation of presynaptic alpha-synuclein is not influenced by its phosphorylation at serine-129.Neurobiol. Dis. 2021; 152: 105291Google Scholar), little is known about how this phosphorylation may affect aSyn's DNA-binding properties. Our previous work suggested that both aSyn and pSyn can bind DNA directly, but that phosphorylation can alter the nature of this interaction in potentially important ways (9Schaser 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. Raber J. Luk K.C. McCullough A.K. Woltjer R.L. Unni V.K. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.Sci. Rep. 2019; 9: 10919Google Scholar). Given that a detailed understanding of how these two synuclein forms interact with DNA is still lacking, here we set out to test the properties of DNA that are important for aSyn binding and how serine-129 phosphorylation could modulate synuclein–DNA interactions. Our previous work has shown that both aSyn and pSyn can bind 300 bp double-stranded DNA, but that aSyn produced multiple bound states, while pSyn produced only a single bound state under the conditions tested (9Schaser 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. Raber J. Luk K.C. McCullough A.K. Woltjer R.L. Unni V.K. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.Sci. Rep. 2019; 9: 10919Google Scholar). To better understand this differential binding of aSyn and pSyn to DNA, we tested the ability of each synuclein to bind DNA of different lengths. We found that both aSyn and pSyn bind double-stranded DNA in a length-dependent manner (Fig. 1A). Increasing concentrations of synuclein incubated with a ladder containing DNA of various lengths and analyzed using a 10% polyacrylamide gel electrophoretic mobility shift assay (EMSA) system demonstrated that both proteins show little obvious interaction with DNA that is ≤200 bp in length, while longer DNAs are bound and shifted upward. Close examination of this shift due to synuclein binding revealed that at a given synuclein concentration, all DNA lengths above a certain critical length were bound and shifted upward, while below this critical length DNA was unbound, and there was no shift in these bands, and the DNA band in between these two lengths (at or near the critical length) exhibited both shifted (bound) and unshifted (unbound) bands (Fig. 1A). The value of these critical lengths, where the DNA exhibited both shifted and unshifted bands, decreased with increasing synuclein concentration. aSyn produced a range of bound states and, therefore, smearing of the signal at higher DNA lengths obscured the changes occurring to specific bands in the aSyn EMSA. This smearing did not occur with pSyn. This is consistent with our previous work showing that aSyn binding to 300 bp DNA produced a laddering effect due to the presence of multiple shifted bound states of different apparent lengths, while pSyn only produced a single shifted bound state (9Schaser 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. Raber J. Luk K.C. McCullough A.K. Woltjer R.L. Unni V.K. Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.Sci. Rep. 2019; 9: 10919Google Scholar). To understand the DNA length dependence of these band shifts better, without the complication of multiple-length DNA fragments in the same lane, we performed similar EMSAs using DNA of only a single length, ranging from 125 to 500 bp. These experiments also showed a clear length dependence to aSyn and pSyn DNA binding, with aSyn binding DNA better than pSyn at DNA lengths between 125 and 300 bp (Fig. 1B). The Hill slopes for binding of aSyn and pSyn were both positive, suggesting cooperative binding interactions between both synuclein forms and DNA, although we did not detect significant differences in the amount of cooperativity between aSyn or pSyn binding DNA. Given the relative simplicity of interpreting mobility changes with pSyn, due to the lack of smearing at high apparent DNA lengths, we also tested the ability of pSyn to shift ladder DNA under different gel conditions. We saw similar upward shifts of all DNA fragments above a certain critical length, no change below this critical length, and partial binding at or near these critical lengths when performing EMSAs with polyacrylamide gel concentrations ranging between 6 and 20% (Fig. S1A). To assay binding of aSyn to 300 bp DNA using another parallel technique to EMSA, we performed atomic force microscopy (AFM) on surfaces where DNA was applied either without aSyn or in the presence of 57 μM aSyn, a concentration where we measure robust aSyn-DNA binding in our EMSA experiments. These AFM data show evidence of increased apparent DNA thickness and a globular protein signal bound to 300 bp DNA in the condition where aSyn is present (Fig. S1B), suggesting that aSyn does bind 300 bp double-stranded DNA, as previous AFM studies have shown with longer DNA molecules (51Jiang 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: 15685-15690Google Scholar). The physiologically and pathologically relevant conformations of aSyn are subject to debate, and questions remain about the role/s of the unfolded monomeric state (59Lashuel H.A. Overk C.R. Oueslati A. Masliah E. The many faces of α-synuclein: From structure and toxicity to therapeutic target.Nat. Rev. Neurosci. 2013; 14: 38-48Google Scholar, 60Fauvet B. Mbefo M.K. Fares M.B. Desobry C. Michael S. Ardah M.T. Tsika E. Coune P. Prudent M. Lion N. Eliezer D. Moore D.J. Schneider B. Aebischer P. El-Agnaf O.M. et al.α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.J. Biol. Chem. 2012; 287: 15345-15364Google Scholar), as well as a tetrameric structure that it can also form (61Bartels T. Choi J.G. Selkoe D.J. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation.Nature. 2011; 477: 107-110Google Scholar). Its ability to bind DNA resides in the N-terminal portion of the protein, and previous studies have noted the importance of electrostatic interactions between positively charged lysine residues in the N-terminal domain and the phosphate groups within