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Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack
DNA Repair 87 (2020) 102804
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Review Article
Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack
T
Roy Anindya Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, 502285, India
ARTICLE INFO
ABSTRACT
Keywords: AlkB ALKBH ALKBH3 RAD51 RAD51C DNA repair ssDNA Abasic site AGT MGMT NEIL3 UNG2
Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication.
1. Introduction The human genomic DNA is exposed to various DNA damaging chemicals. However, different DNA repair pathways protect genomic DNA and ensure cell survival. Although DNA repair pathways dedicated to repairing double-helical DNA are well understood, much less known about the repair of single-stranded DNA (ssDNA). Curiously, biochemical studies suggested that some DNA repair enzymes could preferentially repair single-stranded DNA, but these results have been largely ignored. Recently, cancer whole-genome sequencing and genetic analysis in budding yeast have established that mutation distribution in the genome is not completely random but occur in clusters that correspond to single-stranded regions of the genome [1,2]. This phenomenon is known as 'kataegis' (recently reviewed in Refs. [3,4]). Further, intriguing mechanisms describing how some of the ssDNA repair proteins are accurately recruited to ssDNA regions of the genome have been discovered. This review aims to provide the update on recent findings on the source of DNA damage in ssDNA, how the repair proteins counteract these genotoxic adducts, and what might be the significance of ssDNA repair. 2. Generation of ssDNA The genetic information of eukaryotic organisms is encoded in
double-stranded DNA. However, the formation of ssDNA is necessary during replication, recombination and transcription. If replication fork is stalled due to DNA damage or nucleotide deprivation, DNA-bound proteins, the minichromosome maintenance (MCM) helicase continues unwinding the DNA and generates ssDNA between the helicase and DNA polymerases [5,6]. While the cells with a functional replication checkpoint response can restrict this ssDNA accumulation, the length of ssDNA could be up to a kilobase in checkpoint mutants cells [7]. Long stretches of ssDNA could also be associated with transcription elongation [8]. Transcription generates negative DNA supercoiling behind the translocating RNA polymerase [9]. Negative supercoiling may facilitate the RNA transcript to base pair with its DNA template strand, leaving the non-transcribed strand as a single-stranded bubble, known as Rloop. [10]. Any impediments to RNA polymerase elongation, including head-on collisions with replication apparatus, loss of the DNA relaxing activity of topoisomerase-I, further increases ssDNA formation via Rloop generation [11]. Transcription-associated ssDNA generation also helps the DNA modifying enzymes, such as AID/APOBEC cytidine deaminases. AID (activation-induced cytidine deaminase), which is primarily expressed in germinal center B cells, mediate somatic hypermutation and class switch recombination by deaminating ssDNA within immunoglobulin genes [12] and APOBEC (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like), expressed primarily in hematopoietic cell populations, inhibits retroviruses through
E-mail address: [email protected]. https://doi.org/10.1016/j.dnarep.2020.102804 Received 28 December 2019; Received in revised form 18 January 2020; Accepted 18 January 2020 Available online 20 January 2020 1568-7864/ © 2020 Elsevier B.V. All rights reserved.
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deamination of cytidine residues to uridines in retroviral reverse-transcribed DNA intermediates [13]. Interestingly, recent whole-genome and exome datasets analysis of cancer cells suggests that APOBEC mutation pattern also results in mutation clusters, indicating the role of ssDNA [14]. One of the reasons cancer cells are vulnerable to APOBEC mutagenesis might be an aberrant accumulation of R-loops in cancer cells [15]. Homologous recombination (HR) repair following doublestrand break (DSB) also involves ssDNA generation. The first step in the repair of a DSB by HR is the 5′→3′ degradation of both DNA ends by a process known as DNA end-resection. HR is initiated by MRN (MRE11RAD50-NBS1) protein complex in the presence of CtIP [16]. MRN complex, owing to high affinity for DSB ends, localize to sites of DNA damage. The Rad50 protein is involved in inter-complex associations and DNA tethering via its zinc hook domain [17]. NBS1 protein of the MRN complex interacts and recruits CtIP to DSB. The MRE11 protein of the MRN complex has both endonuclease and exonuclease activity. In the presence of CtIP, the MRE11 endonuclease activity is promoted, and resection is initiated by internal cleavage downstream to the 5′ end of the DSB. Resected is extended by two more nucleases, EXO1 and DNA2 [18]. EXO1 is a 5′→3′ dsDNA exonuclease, whereas, DNA2 has helicase and endonuclease activity. Together, EXO1 and DNA2 produce ssDNA of several kilobases long, suitable for HR. The resulting 3′ ssDNA binds RAD51 and promotes strand alignment and strand exchange [19]. Formation of RAD51 filament is facilitated by BRCA2 and RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 [20]. Because the homology search and strand invasion steps take about 30 min in mammalian cells [21], long stretches of ssDNA must be protected from exposure to clastogenic chemicals.
known as alkylation repair protein-B (AlkB) [33,34]. AlkB is an iron (FeII) and 2-oxoglutarate-dependent dioxygenase and removes the methyl group as formaldehyde, thereby regenerating the unmodified base. From sequence homology searches, it was found that there are nine sequence homologs of AlkB present in the human genome, namely, ALKBH1-8 and FTO (ALKBH9) [35,36]. However, only ALKBH2 and ALKBH3 are true DNA repair enzymes [37]. Analysis of the substrate specificities of ALKBH2 and ALKBH3 revealed that ALKBH3 preferentially repair alkylated ssDNA, whereas ALKBH2 is specific to dsDNA [38,39]. Although it is critical for the cell to protect the ssDNA from chemical damage, locating ssDNA from the vast majority of dsDNA might be difficult for the DNA repair enzymes. Therefore, DNA repair enzymes commonly partner with other proteins for specific targeting. E. coli AlkB protein is known to bind ssDNA binding protein [40–42] and HR repair pathway protein RecA [43]. RecA protein orchestrates HR via forming nucleoprotein filament by binding to the single-stranded resected DNA [44,45]. Subsequently, RecA filaments align with a homologous DNA duplex, followed by strand exchange and branch migration [46]. Interestingly, RecA also recruits AlkB to ssDNA and stimulates AlkB-mediated repair of alkylated ssDNA [43]. A large amount of RecA protein is produced during SOS response, and it might recruit AlkB to keep the resected ssDNA damage-free. During HR repair, human cells rely on recombinase RAD51, BRCA2 and RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 for nucleoprotein filament formation [47]. Recently, it has been shown that ALKBH3 interacts with human RAD51 paralogue RAD51C, and the interaction domain has been mapped to the amino acid residues 33–52 of RAD51C [48]. RAD51C is known to be part of at least three stable complexes; a dimeric complex of RAD51C-XRCC3 and a larger complex composed of RAD51B-C-D-XRCC2 [49]. RAD51C is also formed a complex with Fanconi proteins PALB2 (FANCN) [50]. Interestingly, RAD51C-ALKBH3 interaction does not hinder the interaction of RAD51C with other partner proteins [48]. As RAD51C is associated with stalled RF and resected ssDNA, and ALKBH3 expression is cell cycle-independent [51], the interaction of ALKBH3 with RAD51C might help ALKBH3 to repair lesions from resected ssDNA or single-stranded gaps behind the stalled RFs. Exposure to DNA methylating agents MMS also generates O6-methylguanine (O6meG) and O4-methylthymine (O4meG). Both adduct types are mutagenic, while O6meG is cytotoxic as well [52]. In human cells, O6meG adduct is repaired by the O6-alkylguanine alkyltransferase (AGT, also known as methylguanine methyltransferase, MGMT) [53]. Interestingly, AGT can repair O6meG from ssDNA and dsDNA [54,55]. AGT binds ssDNA in a cooperative manner in which multiple AGT molecules are assembled with ∼4 nucleotides intervals where each AGT molecule occupies ∼8 base pairs of DNA duplex [56]. Because cooperative binding of AGT to ssDNA allows a higher density of protein on the DNA than non-cooperative binding modes, it might enable efficient surveillance and specific ssDNA. As a result, AGT might be recruited to ssDNA without any partner protein. AGT mRNA level remains unaltered during the cell cycle in normal human fibroblasts, suggesting that AGT expression is not regulated by cell cycle [57]. Mammalian cells depend on accurate HR to repair DNA damage. However, recombination occurs between identical or nearly identical sequences and recombination between inappropriate homologous sequence leads to translocations or other deleterious mutations. ALKBH3 mediated repair of N3meC and N1meA from the resected strand might ensure fidelity of recombination. Similarly, AGT can remove O6meG and O4meG from the ssDNA that accumulates upstream of the stalled replication forks, R-loop or resected DNA, as AGT is present throughout the cell cycle and binds ssDNA in a highly specific cooperative manner.
3. RPA-bound ssDNA is not protected from chemical damage The ssDNA is more vulnerable to attack by the nuclease and DNA damaging reactive endogenous and exogenous chemicals than dsDNA due to lack of regular base pairing and base stacking. Therefore, ssDNA regions need to be adequately protected to avoid loss of genetic information. Eukaryotic ssDNA-binding proteins, such as the heterotrimeric replication protein A (RPA) complex, binds to the ssDNA with high affinity and protects the ssDNA from nucleases [22]. RPA also prevents secondary structure formation [23] and participates in DNA damage checkpoint response [24]. It may be asked whether the RPAbound ssDNA still has the risk of getting damaged by the endogenous DNA damaging chemicals. The vulnerability of ssDNA to DNA damaging chemical was proven when ssDNA was shown to accumulate high frequency of closely-spaced multiple mutations, known as kataegis [3]. The hypermutability of ssDNA mutational clusters was also shown in yeast and various cancer genomes [2,4,25]. When the genes needed for the replication fork assembly were mutated, sequence analysis using a read-depth-based algorithm detected clustered pattern of localized mutation in the human cancer genome [26,27]. This is not unexpected because RPA binding to ssDNA being flexible and dynamic, DNA bases are accessible to the solvent [28] and prone to chemical modification. 4. DNA repair enzymes safeguard ssDNA from alkylating agents Ubiquitous intracellular methyl-donor S-Adenosylmethionine (SAM) can act as a chemical alkylating agent, spontaneously and nonenzymatically transferring its methyl group to DNA [29]. The tissuelevel concentration of SAM ranges from 25 to 50 μM, which is equivalent to 10–20 nM of carcinogenic DNA alkylating agent methyl methanesulfonate (MMS) [30]. MMS reacts with the DNA base ring Nitrogens (N) and exocyclic Oxygen (O) atoms to form a variety of covalent adducts depending on whether the DNA is double‐stranded or single‐stranded. Most DNA alkylating agents preferentially modify the N1‐position of adenine and the N3‐position of cytosine only in ssDNA as these sites are engaged in base pairing in double‐stranded DNA (reviewed in Ref [31,32]). The E. coli enzyme that repairs these adducts is
5. Removal of uracil from ssDNA by DNA repair enzymes Uracil, being structurally similar to thymine, occasionally misincorporated into DNA by DNA polymerases when the cellular dUTP 2
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level is high. Such conditions might arise if folic acid is deficient in the diet [58] or thymidylate synthase Inhibitors are used as cancer chemotherapy drugs [59]. Uracil in DNA might also originate spontaneously by hydrolytic deamination of cytosine. Interestingly, ssDNA is more prone to spontaneous deamination than duplex DNA [60]. Deamination of cytosine to uracil (C-to-U) in ssDNA might also be catalyzed by homologs of AID/APOBEC family of cytidine deaminases. Aberrant overexpression of APOBEC activity may be a major source of mutagenesis in human cancers [2]. Significantly, APOBEC family members, APOBEC3A and APOBEC3B, specifically deaminates cytosine in ssDNA [61,62]. A study focusing on a mutation introduced at a specific cytosine residue in the lacZα gene showed that this cytosine was deaminated 100-fold more slowly in E. coli double-stranded DNA genome [63], than in bacteriophage M13 [64] ssDNA genome. Uracil in the genome is usually excised by uracil-DNA glycosylase (UDG) [65], a family of monofunctional glycosylase lacking AP-lyase activity. Mammalian cells express five UDG enzymes, namely, UNG1, UNG2, SMUG1, TDG and MBD4. UNG splice-variants, UNG1 is localized in mitochondria and UNG2 is nuclear [66]. Although UNG1 and UNG2 share an identical catalytic domain, only UNG2 excises uracil from ssDNA [67]. By binding to RPA and proliferating cell nuclear antigen (PCNA), UNG2 can be localized in the vicinity of replication forks [68]. Interestingly, a genetic experiment in yeast showed that most of the APOBEC-induced abasic sites were bypassed in an error-free manner during replication [69]. Another UDG family protein SMUG1 (single-strand-selective monofunctional UDG) excises uracil from ssDNA, albeit with much lower efficiency than UNG2, but act on a variety of substrates, including thymine oxidation product 5-hydroxymethyl uracil. [67]. Initially, SMUG1 was reported to be selective for ssDNA [70], but later it was found that SMUG1 repairs uracil-containing double-stranded DNA substrates [71].
FapyG and FapyA [82]. NEIL3 expression is known to be cell cycledependent and present in the S phase and G2 phases only [85,86]. Because of its specificity for ssDNA, a wide range of substrates, S phasespecific expression and nuclear localization, NEIL3 might be an important enzyme involved in repairing oxidative damage from ssDNA during replication. 7. Processing of abasic sites in ssDNA Replication fork-stalling causes an accumulation of ssDNA due to the uncoupling of DNA synthesis from DNA unwinding [87]. If cytosine deamination within this ssDNA forms uracil or 8-oxoG oxidation products (Sp, Gh) accumulates, then the damaged base could be excised by UNG2 or NEIL3, respectively. In either case, the glycosylase-mediated excision would create abasic sites before the restart of DNA replication. Indeed, the accumulation of clusters of abasic sites was shown to form in the replicated DNA in response to oxidative damage [88]. Why would repair enzyme create abasic site in ssDNA when the abasic sites in ssDNA might result in single-strand breaks (SSBs)? Abasic sites are chemically unstable and have a very short half-life, especially at the mild alkaline condition. The deoxyribose at abasic sites are electrophilic and undergo a spontaneous reversible reaction to form hemiacetal and the open-chain aldehyde forms that are highly reactive and lead to strand break [89]. Previous studies have demonstrated that AP endonuclease APE1, can cleave the abasic sites present in ssDNA, RNA/ DNA hybrid, and RNA besides normal duplex DNA [65,90]. Thus, by creating an abasic site on ssDNA at the stalled replication fork, UNG2 or NEIL3 glycosylase most likely generate SSBs. SSBs are rather common lesions arising in cells and repaired efficiently (recently reviewed in Ref [91].). Most SSBs are detected by SSB sensor protein PARP1 and processed within minutes by a pathway that is promoted by an ensemble of proteins, including the molecular scaffold protein XRCC1, polynucleotide kinase/phosphatase PNKP, 3′-end processing enzyme TDP1, DNA ligase LIG3 [92]. Given the role of XRCC1 in processing SSBs [93], it is possible that ssDNA repair enzymes coordinate with XRCC1 to play a significant role in the S phase of the cell cycle. However, more studies are needed to establish the role of ssDNA-specific repair in maintaining genome integrity.
6. Removal of oxidative damages from ssDNA Reactive oxygen species (ROS) are produced as byproducts of respiration and synthesized during the inflammatory response. Exposure to ROS results cause oxidation damage of the DNA, and the most common oxidation product is known as 8-oxoguanine (8-oxoG) [72], which readily oxidizes to one-electron oxidation products including potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5formamidopyrimidine (FapyA), thymine glycol (5,6-dihydro-5,6-dihydroxythymine), 5-hydroxycytosine (5-OHC) and its deamination products, 5-hydroxyuracil (5-OHU). Number of DNA glycosylases have been identified in mammalian cells which can repair oxidatively damaged bases, for example, 8-oxoguanine-DNA glycosylase-1 (OGG1), endonuclease three homolog-1 (NTH1) and Nei-like (NEIL) glycosylases. OGG1 is the key repair enzyme for 8-oxoG in mammals and NTH1 has a minor supportive role in 8-oxoG removal, but its primary substrate is thymine glycol, 5-OHC and 5-OHU [73]. OGG1 and NTH1 also repair FapyG and FapyA, respectively [74]. There are three Nei-like (NEIL) DNA glycosylases in human cells (NEIL1-3); among these, NEIL1 and NEIL2 are bifunctional as they also have 3′ AP-lyase activity [75,76]. Both NEIL1 and NEIL2 repair 8-oxoG oxidation products Gh and Sp [77]. While NEIL1 excises FapyA, FapyG, thymine glycol, and 5-OHU, NEIL2 prefers 5-OHU (17–18). OGG1 and NTH1 prefer double-stranded DNA, NEIL1 and NEIL2 have a preference for partly double-stranded bubble structure [78]. Among the three NEIL glycosylases, only NEIL3 repairs damaged ssDNA [79], whereas, OGG1, NTH1, NEIL1 and NEIL2 completely lacks ssDNA-specific activity [78,80,81]. NEIL3 is localized in the nucleus and have a PCNA-binding motif commonly found in PCNAbinding proteins [82]. The co-localization experiment showed recombinant EGFP fused NEIL3 co-localizes with RPA [83]. RPA interaction might increase molecular crowding nearby the ssDNA regions and facilitate the processive searching (reviewed in ref [84]). NEIL3 also has broad substrate specificity, including Sp, Gh, Tg,
8. Future perspective One of the reasons why ssDNA damage repair is under-recognized is difficulty in the detection of ssDNA in vivo. The majority of the typical DNA dyes are intercalating agents and cannot be used for visualization of ssDNA. Hence, ssDNA can be detected by bromo-deoxyuridine (BrdU) incorporation and immunofluorescence staining using an antiBrdU antibody [94]. Recently identified ultrafine anaphase bridges (UFB), which might have some ssDNA, are not always detectable by the DNA dyes. Such UFBs could be detected by immunofluorescence, using antibodies against UFB-associated factors, including RPA [95]. Viruses having ssDNA genome, such as parvoviruses, undergo rolling circle replication in the nucleus [96,97]. The parvoviruses are known to induce DNA damage response [98,99] and might offer valuable insight into the extent of ssDNA repair in mammalian cells. Besides, parvoviruses ssDNA genome contains unique hairpin structures at the 5′ and 3′ ends, which may allow its specific detection inside the nucleus. Reactivation of parvoviruses inactivated by alkylation or uridine incorporation might provide direct evidence for ssDNA repair. Recently, imaging techniques involving micro-irradiation could now be applied to the study of base excision repair and single-strand break repair (reviewed in Ref [100].). It is expected that the application of micro-irradiation to study ssDNA base damage repair might yield new insights. The single-molecule imaging technique, such as ssDNA curtain assay [101], can be employed to unveil the mechanism of ssDNA repair. Future studies should also try to address if the ssDNA repair proteins resort to the processive searching mechanism by interacting with other 3
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Table 1 ssDNA repair proteins. Protein
Type of DNA damage repaired
Type of ssDNA
Mode of recruitment of ssDNA
ALKBH3 AGT UNG2 NEIL3
N3meC, N1meA O6meG, O4meT Uracil Sp, Gh, Tg, FapyG, FapyA
Resected ssDNA Replication fork Replication fork Replication fork
interaction with RAD51C Cooperative binding interaction with RPA1, PCNA interaction with RPA1, PCNA
repair proteins to increase molecular crowding surrounding the ssDNA regions.
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9. Conclusion Removal of adducts from the ssDNA could be a critical repair mechanism for protecting the integrity of ssDNA. As our understanding of key players of ssDNA continues to advance, a general overview of the different ssDNA repair mechanisms and how they to function are provided here (summarized in Table 1). Since the frequency of spontaneous alkylation, hydrolysis and oxidation are significantly higher in ssDNA, the role of ALKBH3, AGT, UNG2 and NEIL3 in ssDNA repair might not be redundant. Alkylation damage repair in ssDNA generated upstream of stalled replication fork can facilitate replication and prevent fork-stalling; excision of oxidatively damaged base or uracil by DNA glycosylases from ssDNA generates single-strand breaks leading to collapsed replication forks. It is not known whether damage removal from ssDNA undermines the trans-lesion synthesis by Y-family DNA polymerases. Our understanding of ssDNA repair also remains limited with respect to the poorly defined phenotypes of defects of ssDNA repair, the tissue-specific capacity of ssDNA repair, mitochondrial ssDNA repair, and the role of ssDNA repair in aging. Accumulating evidence suggests that ssDNA repair might be critical to the viability of replicating cells. Further study is required to completely understand how ssDNA repair contributes to genome integrity and protection from chemical damage. Funding This work was supported by the Science and Engineering Research Board (SERB), Govt. of India (EMR/2016/005135/BBM). Declaration of Competing Interest The author declares that there are no conflicts of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.dnarep.2020.102804. References [1] S. Jinks-Robertson, K. Chan, J.F. Sterling, S.A. Roberts, A.S. Bhagwat, M.A. Resnick, D.A. Gordenin, Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent, PLoS Genet. 8 (2012). [2] Steven A. Roberts, J. Sterling, C. Thompson, S. Harris, D. Mav, R. Shah, Leszek J. Klimczak, Gregory V. Kryukov, E. Malc, Piotr A. Mieczkowski, Michael A. Resnick, Dmitry A. Gordenin, Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions, Mol. Cell 46 (2012) 424–435. [3] K. Chan, D.A. Gordenin, Clusters of multiple mutations: incidence and molecular mechanisms, Annu. Rev. Genet. 49 (2015) 243–267. [4] S.A. Roberts, D.A. Gordenin, Hypermutation in human cancer genomes: footprints and mechanisms, Nat. Rev. Cancer 14 (2014) 786–800. [5] C. Van, S. Yan, W.M. Michael, S. Waga, K.A. Cimprich, Continued primer synthesis at stalled replication forks contributes to checkpoint activation, J. Cell Biol. 189 (2010) 233–246.
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Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Review Article
Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack
T
Roy Anindya Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, 502285, India
ARTICLE INFO
ABSTRACT
Keywords: AlkB ALKBH ALKBH3 RAD51 RAD51C DNA repair ssDNA Abasic site AGT MGMT NEIL3 UNG2
Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication.
1. Introduction The human genomic DNA is exposed to various DNA damaging chemicals. However, different DNA repair pathways protect genomic DNA and ensure cell survival. Although DNA repair pathways dedicated to repairing double-helical DNA are well understood, much less known about the repair of single-stranded DNA (ssDNA). Curiously, biochemical studies suggested that some DNA repair enzymes could preferentially repair single-stranded DNA, but these results have been largely ignored. Recently, cancer whole-genome sequencing and genetic analysis in budding yeast have established that mutation distribution in the genome is not completely random but occur in clusters that correspond to single-stranded regions of the genome [1,2]. This phenomenon is known as 'kataegis' (recently reviewed in Refs. [3,4]). Further, intriguing mechanisms describing how some of the ssDNA repair proteins are accurately recruited to ssDNA regions of the genome have been discovered. This review aims to provide the update on recent findings on the source of DNA damage in ssDNA, how the repair proteins counteract these genotoxic adducts, and what might be the significance of ssDNA repair. 2. Generation of ssDNA The genetic information of eukaryotic organisms is encoded in
double-stranded DNA. However, the formation of ssDNA is necessary during replication, recombination and transcription. If replication fork is stalled due to DNA damage or nucleotide deprivation, DNA-bound proteins, the minichromosome maintenance (MCM) helicase continues unwinding the DNA and generates ssDNA between the helicase and DNA polymerases [5,6]. While the cells with a functional replication checkpoint response can restrict this ssDNA accumulation, the length of ssDNA could be up to a kilobase in checkpoint mutants cells [7]. Long stretches of ssDNA could also be associated with transcription elongation [8]. Transcription generates negative DNA supercoiling behind the translocating RNA polymerase [9]. Negative supercoiling may facilitate the RNA transcript to base pair with its DNA template strand, leaving the non-transcribed strand as a single-stranded bubble, known as Rloop. [10]. Any impediments to RNA polymerase elongation, including head-on collisions with replication apparatus, loss of the DNA relaxing activity of topoisomerase-I, further increases ssDNA formation via Rloop generation [11]. Transcription-associated ssDNA generation also helps the DNA modifying enzymes, such as AID/APOBEC cytidine deaminases. AID (activation-induced cytidine deaminase), which is primarily expressed in germinal center B cells, mediate somatic hypermutation and class switch recombination by deaminating ssDNA within immunoglobulin genes [12] and APOBEC (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like), expressed primarily in hematopoietic cell populations, inhibits retroviruses through
E-mail address: [email protected]. https://doi.org/10.1016/j.dnarep.2020.102804 Received 28 December 2019; Received in revised form 18 January 2020; Accepted 18 January 2020 Available online 20 January 2020 1568-7864/ © 2020 Elsevier B.V. All rights reserved.
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deamination of cytidine residues to uridines in retroviral reverse-transcribed DNA intermediates [13]. Interestingly, recent whole-genome and exome datasets analysis of cancer cells suggests that APOBEC mutation pattern also results in mutation clusters, indicating the role of ssDNA [14]. One of the reasons cancer cells are vulnerable to APOBEC mutagenesis might be an aberrant accumulation of R-loops in cancer cells [15]. Homologous recombination (HR) repair following doublestrand break (DSB) also involves ssDNA generation. The first step in the repair of a DSB by HR is the 5′→3′ degradation of both DNA ends by a process known as DNA end-resection. HR is initiated by MRN (MRE11RAD50-NBS1) protein complex in the presence of CtIP [16]. MRN complex, owing to high affinity for DSB ends, localize to sites of DNA damage. The Rad50 protein is involved in inter-complex associations and DNA tethering via its zinc hook domain [17]. NBS1 protein of the MRN complex interacts and recruits CtIP to DSB. The MRE11 protein of the MRN complex has both endonuclease and exonuclease activity. In the presence of CtIP, the MRE11 endonuclease activity is promoted, and resection is initiated by internal cleavage downstream to the 5′ end of the DSB. Resected is extended by two more nucleases, EXO1 and DNA2 [18]. EXO1 is a 5′→3′ dsDNA exonuclease, whereas, DNA2 has helicase and endonuclease activity. Together, EXO1 and DNA2 produce ssDNA of several kilobases long, suitable for HR. The resulting 3′ ssDNA binds RAD51 and promotes strand alignment and strand exchange [19]. Formation of RAD51 filament is facilitated by BRCA2 and RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 [20]. Because the homology search and strand invasion steps take about 30 min in mammalian cells [21], long stretches of ssDNA must be protected from exposure to clastogenic chemicals.
known as alkylation repair protein-B (AlkB) [33,34]. AlkB is an iron (FeII) and 2-oxoglutarate-dependent dioxygenase and removes the methyl group as formaldehyde, thereby regenerating the unmodified base. From sequence homology searches, it was found that there are nine sequence homologs of AlkB present in the human genome, namely, ALKBH1-8 and FTO (ALKBH9) [35,36]. However, only ALKBH2 and ALKBH3 are true DNA repair enzymes [37]. Analysis of the substrate specificities of ALKBH2 and ALKBH3 revealed that ALKBH3 preferentially repair alkylated ssDNA, whereas ALKBH2 is specific to dsDNA [38,39]. Although it is critical for the cell to protect the ssDNA from chemical damage, locating ssDNA from the vast majority of dsDNA might be difficult for the DNA repair enzymes. Therefore, DNA repair enzymes commonly partner with other proteins for specific targeting. E. coli AlkB protein is known to bind ssDNA binding protein [40–42] and HR repair pathway protein RecA [43]. RecA protein orchestrates HR via forming nucleoprotein filament by binding to the single-stranded resected DNA [44,45]. Subsequently, RecA filaments align with a homologous DNA duplex, followed by strand exchange and branch migration [46]. Interestingly, RecA also recruits AlkB to ssDNA and stimulates AlkB-mediated repair of alkylated ssDNA [43]. A large amount of RecA protein is produced during SOS response, and it might recruit AlkB to keep the resected ssDNA damage-free. During HR repair, human cells rely on recombinase RAD51, BRCA2 and RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 for nucleoprotein filament formation [47]. Recently, it has been shown that ALKBH3 interacts with human RAD51 paralogue RAD51C, and the interaction domain has been mapped to the amino acid residues 33–52 of RAD51C [48]. RAD51C is known to be part of at least three stable complexes; a dimeric complex of RAD51C-XRCC3 and a larger complex composed of RAD51B-C-D-XRCC2 [49]. RAD51C is also formed a complex with Fanconi proteins PALB2 (FANCN) [50]. Interestingly, RAD51C-ALKBH3 interaction does not hinder the interaction of RAD51C with other partner proteins [48]. As RAD51C is associated with stalled RF and resected ssDNA, and ALKBH3 expression is cell cycle-independent [51], the interaction of ALKBH3 with RAD51C might help ALKBH3 to repair lesions from resected ssDNA or single-stranded gaps behind the stalled RFs. Exposure to DNA methylating agents MMS also generates O6-methylguanine (O6meG) and O4-methylthymine (O4meG). Both adduct types are mutagenic, while O6meG is cytotoxic as well [52]. In human cells, O6meG adduct is repaired by the O6-alkylguanine alkyltransferase (AGT, also known as methylguanine methyltransferase, MGMT) [53]. Interestingly, AGT can repair O6meG from ssDNA and dsDNA [54,55]. AGT binds ssDNA in a cooperative manner in which multiple AGT molecules are assembled with ∼4 nucleotides intervals where each AGT molecule occupies ∼8 base pairs of DNA duplex [56]. Because cooperative binding of AGT to ssDNA allows a higher density of protein on the DNA than non-cooperative binding modes, it might enable efficient surveillance and specific ssDNA. As a result, AGT might be recruited to ssDNA without any partner protein. AGT mRNA level remains unaltered during the cell cycle in normal human fibroblasts, suggesting that AGT expression is not regulated by cell cycle [57]. Mammalian cells depend on accurate HR to repair DNA damage. However, recombination occurs between identical or nearly identical sequences and recombination between inappropriate homologous sequence leads to translocations or other deleterious mutations. ALKBH3 mediated repair of N3meC and N1meA from the resected strand might ensure fidelity of recombination. Similarly, AGT can remove O6meG and O4meG from the ssDNA that accumulates upstream of the stalled replication forks, R-loop or resected DNA, as AGT is present throughout the cell cycle and binds ssDNA in a highly specific cooperative manner.
3. RPA-bound ssDNA is not protected from chemical damage The ssDNA is more vulnerable to attack by the nuclease and DNA damaging reactive endogenous and exogenous chemicals than dsDNA due to lack of regular base pairing and base stacking. Therefore, ssDNA regions need to be adequately protected to avoid loss of genetic information. Eukaryotic ssDNA-binding proteins, such as the heterotrimeric replication protein A (RPA) complex, binds to the ssDNA with high affinity and protects the ssDNA from nucleases [22]. RPA also prevents secondary structure formation [23] and participates in DNA damage checkpoint response [24]. It may be asked whether the RPAbound ssDNA still has the risk of getting damaged by the endogenous DNA damaging chemicals. The vulnerability of ssDNA to DNA damaging chemical was proven when ssDNA was shown to accumulate high frequency of closely-spaced multiple mutations, known as kataegis [3]. The hypermutability of ssDNA mutational clusters was also shown in yeast and various cancer genomes [2,4,25]. When the genes needed for the replication fork assembly were mutated, sequence analysis using a read-depth-based algorithm detected clustered pattern of localized mutation in the human cancer genome [26,27]. This is not unexpected because RPA binding to ssDNA being flexible and dynamic, DNA bases are accessible to the solvent [28] and prone to chemical modification. 4. DNA repair enzymes safeguard ssDNA from alkylating agents Ubiquitous intracellular methyl-donor S-Adenosylmethionine (SAM) can act as a chemical alkylating agent, spontaneously and nonenzymatically transferring its methyl group to DNA [29]. The tissuelevel concentration of SAM ranges from 25 to 50 μM, which is equivalent to 10–20 nM of carcinogenic DNA alkylating agent methyl methanesulfonate (MMS) [30]. MMS reacts with the DNA base ring Nitrogens (N) and exocyclic Oxygen (O) atoms to form a variety of covalent adducts depending on whether the DNA is double‐stranded or single‐stranded. Most DNA alkylating agents preferentially modify the N1‐position of adenine and the N3‐position of cytosine only in ssDNA as these sites are engaged in base pairing in double‐stranded DNA (reviewed in Ref [31,32]). The E. coli enzyme that repairs these adducts is
5. Removal of uracil from ssDNA by DNA repair enzymes Uracil, being structurally similar to thymine, occasionally misincorporated into DNA by DNA polymerases when the cellular dUTP 2
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level is high. Such conditions might arise if folic acid is deficient in the diet [58] or thymidylate synthase Inhibitors are used as cancer chemotherapy drugs [59]. Uracil in DNA might also originate spontaneously by hydrolytic deamination of cytosine. Interestingly, ssDNA is more prone to spontaneous deamination than duplex DNA [60]. Deamination of cytosine to uracil (C-to-U) in ssDNA might also be catalyzed by homologs of AID/APOBEC family of cytidine deaminases. Aberrant overexpression of APOBEC activity may be a major source of mutagenesis in human cancers [2]. Significantly, APOBEC family members, APOBEC3A and APOBEC3B, specifically deaminates cytosine in ssDNA [61,62]. A study focusing on a mutation introduced at a specific cytosine residue in the lacZα gene showed that this cytosine was deaminated 100-fold more slowly in E. coli double-stranded DNA genome [63], than in bacteriophage M13 [64] ssDNA genome. Uracil in the genome is usually excised by uracil-DNA glycosylase (UDG) [65], a family of monofunctional glycosylase lacking AP-lyase activity. Mammalian cells express five UDG enzymes, namely, UNG1, UNG2, SMUG1, TDG and MBD4. UNG splice-variants, UNG1 is localized in mitochondria and UNG2 is nuclear [66]. Although UNG1 and UNG2 share an identical catalytic domain, only UNG2 excises uracil from ssDNA [67]. By binding to RPA and proliferating cell nuclear antigen (PCNA), UNG2 can be localized in the vicinity of replication forks [68]. Interestingly, a genetic experiment in yeast showed that most of the APOBEC-induced abasic sites were bypassed in an error-free manner during replication [69]. Another UDG family protein SMUG1 (single-strand-selective monofunctional UDG) excises uracil from ssDNA, albeit with much lower efficiency than UNG2, but act on a variety of substrates, including thymine oxidation product 5-hydroxymethyl uracil. [67]. Initially, SMUG1 was reported to be selective for ssDNA [70], but later it was found that SMUG1 repairs uracil-containing double-stranded DNA substrates [71].
FapyG and FapyA [82]. NEIL3 expression is known to be cell cycledependent and present in the S phase and G2 phases only [85,86]. Because of its specificity for ssDNA, a wide range of substrates, S phasespecific expression and nuclear localization, NEIL3 might be an important enzyme involved in repairing oxidative damage from ssDNA during replication. 7. Processing of abasic sites in ssDNA Replication fork-stalling causes an accumulation of ssDNA due to the uncoupling of DNA synthesis from DNA unwinding [87]. If cytosine deamination within this ssDNA forms uracil or 8-oxoG oxidation products (Sp, Gh) accumulates, then the damaged base could be excised by UNG2 or NEIL3, respectively. In either case, the glycosylase-mediated excision would create abasic sites before the restart of DNA replication. Indeed, the accumulation of clusters of abasic sites was shown to form in the replicated DNA in response to oxidative damage [88]. Why would repair enzyme create abasic site in ssDNA when the abasic sites in ssDNA might result in single-strand breaks (SSBs)? Abasic sites are chemically unstable and have a very short half-life, especially at the mild alkaline condition. The deoxyribose at abasic sites are electrophilic and undergo a spontaneous reversible reaction to form hemiacetal and the open-chain aldehyde forms that are highly reactive and lead to strand break [89]. Previous studies have demonstrated that AP endonuclease APE1, can cleave the abasic sites present in ssDNA, RNA/ DNA hybrid, and RNA besides normal duplex DNA [65,90]. Thus, by creating an abasic site on ssDNA at the stalled replication fork, UNG2 or NEIL3 glycosylase most likely generate SSBs. SSBs are rather common lesions arising in cells and repaired efficiently (recently reviewed in Ref [91].). Most SSBs are detected by SSB sensor protein PARP1 and processed within minutes by a pathway that is promoted by an ensemble of proteins, including the molecular scaffold protein XRCC1, polynucleotide kinase/phosphatase PNKP, 3′-end processing enzyme TDP1, DNA ligase LIG3 [92]. Given the role of XRCC1 in processing SSBs [93], it is possible that ssDNA repair enzymes coordinate with XRCC1 to play a significant role in the S phase of the cell cycle. However, more studies are needed to establish the role of ssDNA-specific repair in maintaining genome integrity.
6. Removal of oxidative damages from ssDNA Reactive oxygen species (ROS) are produced as byproducts of respiration and synthesized during the inflammatory response. Exposure to ROS results cause oxidation damage of the DNA, and the most common oxidation product is known as 8-oxoguanine (8-oxoG) [72], which readily oxidizes to one-electron oxidation products including potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5formamidopyrimidine (FapyA), thymine glycol (5,6-dihydro-5,6-dihydroxythymine), 5-hydroxycytosine (5-OHC) and its deamination products, 5-hydroxyuracil (5-OHU). Number of DNA glycosylases have been identified in mammalian cells which can repair oxidatively damaged bases, for example, 8-oxoguanine-DNA glycosylase-1 (OGG1), endonuclease three homolog-1 (NTH1) and Nei-like (NEIL) glycosylases. OGG1 is the key repair enzyme for 8-oxoG in mammals and NTH1 has a minor supportive role in 8-oxoG removal, but its primary substrate is thymine glycol, 5-OHC and 5-OHU [73]. OGG1 and NTH1 also repair FapyG and FapyA, respectively [74]. There are three Nei-like (NEIL) DNA glycosylases in human cells (NEIL1-3); among these, NEIL1 and NEIL2 are bifunctional as they also have 3′ AP-lyase activity [75,76]. Both NEIL1 and NEIL2 repair 8-oxoG oxidation products Gh and Sp [77]. While NEIL1 excises FapyA, FapyG, thymine glycol, and 5-OHU, NEIL2 prefers 5-OHU (17–18). OGG1 and NTH1 prefer double-stranded DNA, NEIL1 and NEIL2 have a preference for partly double-stranded bubble structure [78]. Among the three NEIL glycosylases, only NEIL3 repairs damaged ssDNA [79], whereas, OGG1, NTH1, NEIL1 and NEIL2 completely lacks ssDNA-specific activity [78,80,81]. NEIL3 is localized in the nucleus and have a PCNA-binding motif commonly found in PCNAbinding proteins [82]. The co-localization experiment showed recombinant EGFP fused NEIL3 co-localizes with RPA [83]. RPA interaction might increase molecular crowding nearby the ssDNA regions and facilitate the processive searching (reviewed in ref [84]). NEIL3 also has broad substrate specificity, including Sp, Gh, Tg,
8. Future perspective One of the reasons why ssDNA damage repair is under-recognized is difficulty in the detection of ssDNA in vivo. The majority of the typical DNA dyes are intercalating agents and cannot be used for visualization of ssDNA. Hence, ssDNA can be detected by bromo-deoxyuridine (BrdU) incorporation and immunofluorescence staining using an antiBrdU antibody [94]. Recently identified ultrafine anaphase bridges (UFB), which might have some ssDNA, are not always detectable by the DNA dyes. Such UFBs could be detected by immunofluorescence, using antibodies against UFB-associated factors, including RPA [95]. Viruses having ssDNA genome, such as parvoviruses, undergo rolling circle replication in the nucleus [96,97]. The parvoviruses are known to induce DNA damage response [98,99] and might offer valuable insight into the extent of ssDNA repair in mammalian cells. Besides, parvoviruses ssDNA genome contains unique hairpin structures at the 5′ and 3′ ends, which may allow its specific detection inside the nucleus. Reactivation of parvoviruses inactivated by alkylation or uridine incorporation might provide direct evidence for ssDNA repair. Recently, imaging techniques involving micro-irradiation could now be applied to the study of base excision repair and single-strand break repair (reviewed in Ref [100].). It is expected that the application of micro-irradiation to study ssDNA base damage repair might yield new insights. The single-molecule imaging technique, such as ssDNA curtain assay [101], can be employed to unveil the mechanism of ssDNA repair. Future studies should also try to address if the ssDNA repair proteins resort to the processive searching mechanism by interacting with other 3
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Table 1 ssDNA repair proteins. Protein
Type of DNA damage repaired
Type of ssDNA
Mode of recruitment of ssDNA
ALKBH3 AGT UNG2 NEIL3
N3meC, N1meA O6meG, O4meT Uracil Sp, Gh, Tg, FapyG, FapyA
Resected ssDNA Replication fork Replication fork Replication fork
interaction with RAD51C Cooperative binding interaction with RPA1, PCNA interaction with RPA1, PCNA
repair proteins to increase molecular crowding surrounding the ssDNA regions.
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9. Conclusion Removal of adducts from the ssDNA could be a critical repair mechanism for protecting the integrity of ssDNA. As our understanding of key players of ssDNA continues to advance, a general overview of the different ssDNA repair mechanisms and how they to function are provided here (summarized in Table 1). Since the frequency of spontaneous alkylation, hydrolysis and oxidation are significantly higher in ssDNA, the role of ALKBH3, AGT, UNG2 and NEIL3 in ssDNA repair might not be redundant. Alkylation damage repair in ssDNA generated upstream of stalled replication fork can facilitate replication and prevent fork-stalling; excision of oxidatively damaged base or uracil by DNA glycosylases from ssDNA generates single-strand breaks leading to collapsed replication forks. It is not known whether damage removal from ssDNA undermines the trans-lesion synthesis by Y-family DNA polymerases. Our understanding of ssDNA repair also remains limited with respect to the poorly defined phenotypes of defects of ssDNA repair, the tissue-specific capacity of ssDNA repair, mitochondrial ssDNA repair, and the role of ssDNA repair in aging. Accumulating evidence suggests that ssDNA repair might be critical to the viability of replicating cells. Further study is required to completely understand how ssDNA repair contributes to genome integrity and protection from chemical damage. Funding This work was supported by the Science and Engineering Research Board (SERB), Govt. of India (EMR/2016/005135/BBM). Declaration of Competing Interest The author declares that there are no conflicts of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.dnarep.2020.102804. References [1] S. Jinks-Robertson, K. Chan, J.F. Sterling, S.A. Roberts, A.S. Bhagwat, M.A. Resnick, D.A. Gordenin, Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent, PLoS Genet. 8 (2012). [2] Steven A. Roberts, J. Sterling, C. Thompson, S. Harris, D. Mav, R. Shah, Leszek J. Klimczak, Gregory V. Kryukov, E. Malc, Piotr A. Mieczkowski, Michael A. Resnick, Dmitry A. Gordenin, Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions, Mol. Cell 46 (2012) 424–435. [3] K. Chan, D.A. Gordenin, Clusters of multiple mutations: incidence and molecular mechanisms, Annu. Rev. Genet. 49 (2015) 243–267. [4] S.A. Roberts, D.A. Gordenin, Hypermutation in human cancer genomes: footprints and mechanisms, Nat. Rev. Cancer 14 (2014) 786–800. [5] C. Van, S. Yan, W.M. Michael, S. Waga, K.A. Cimprich, Continued primer synthesis at stalled replication forks contributes to checkpoint activation, J. Cell Biol. 189 (2010) 233–246.
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