Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response

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Review

Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response Laura V. Croft a,1 , Emma Bolderson a,1 , Mark N. Adams a,1 , Serene El-Kamand b,1 , Ruvini Kariawasam b , Liza Cubeddu b,c,∗ , Roland Gamsjaeger b,c,∗ , Derek J. Richard a,∗∗ a Genome Stability Laboratory, Cancer and Ageing Research Program, Institute of Health and Biomedical Innovation, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland 4102, Australia b School of Science and Health, Western Sydney University, Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia c School of Life and Environmental Sciences, University of Sydney, NSW 2006, Australia

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Article history: Received 15 December 2017 Received in revised form 21 March 2018 Accepted 22 March 2018 Available online xxx Keywords: hSSB1 DNA repair Replication fork restart Cell cycle checkpoint activation ssDNA interactions

a b s t r a c t Our genomic DNA is found predominantly in a double-stranded helical conformation. However, there are a number of cellular transactions and DNA damage events that result in the exposure of single stranded regions of DNA. DNA transactions require these regions of single stranded DNA, but they are only transient in nature as they are particularly susceptible to further damage through chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. To protect these exposed regions of single stranded DNA, all living organisms have members of the Single Stranded DNA Binding (SSB) protein family, which are characterised by a conserved oligonucleotide/oligosaccharide-binding (OB) domain. In humans, three such proteins members have been identified; namely the Replication Protein A (RPA) complex, hSSB1 and hSSB2. While RPA is extremely well characterised, the roles of hSSB1 and hSSB2 have only emerged recently. In this review, we discuss the critical roles that hSSB1 plays in the maintenance of genomic stability. © 2018 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Homologous recombination (HR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The role of hSSB1 in the repair of stalled and collapsed replication forks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The role of hSSB1 in the base excision repair (BER) pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Regulation of hSSB1 by INTS3 and their role in transcription termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 hSSB1 and cell cycle regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Structural aspects of hSSB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviations: SSB, single-stranded DNA binding proteins; OB, oligonucleotide/oligosaccharide binding domain; DSB, double-strand DNA break repair; HR, homologous recombination; BER, base excision repair; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; RPA, replication protein A; NHEJ, non-homologous end joining. ∗ Corresponding author at: School of Science and Health, Western Sydney University, Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia. ∗∗ Corresponding author at: Genome Stability Laboratory, Cancer and Ageing Research Program, Institute of Health and Biomedical Innovation, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland 4102, Australia. E-mail addresses: [email protected] (L.V. Croft), [email protected] (E. Bolderson), [email protected] (M.N. Adams), [email protected] (S. El-Kamand), [email protected] (R. Kariawasam), [email protected] (L. Cubeddu), [email protected] (R. Gamsjaeger), [email protected] (D.J. Richard). 1 Contributed equally. https://doi.org/10.1016/j.semcdb.2018.03.014 1084-9521/© 2018 Elsevier Ltd. All rights reserved.

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2

1. Introduction

2. Homologous recombination (HR)

The DNA within our cells exists predominantly in a double stranded DNA (dsDNA) helix. This structure affords a degree of protection for the genetic code. However, the coding bases lie within the helix and must be exposed in normal cellular processes such as DNA replication and transcription. Further, single stranded DNA (ssDNA) can be generated by DNA damaging events, and in normal repair processes ssDNA is required for the repair of DNA damage. Exposed ssDNA poses a real problem for the cell, as damage of this DNA by chemicals and enzymes can result in genomic instability that is difficult to repair, thus it is important that this DNA is quickly sequestered and protected. Underlying the importance of this process, evolution has dictated that all life has no less than one member of the single-stranded DNA binding (SSB) protein family [1–4]. This family is characterised by the presence of at least one structurally conserved oligonucleotide/oligosaccharidebinding (OB) domain. The OB domain, while predominantly being involved in binding to ssDNA, has also evolved to mediate proteinprotein interactions. The SSB family can be further divided into simple and complex (Replication Protein A (RPA)-like) SSBs. The simple SSBs are represented in all domains of life while the more complex RPA-like members are only found in some Archaea and all eukaryotes. The simple SSBs are characterised by a polypeptide with a single OB domain. While containing only a single OB domain in their polypeptide chain, the majority of simple SSBs, including the human versions, do however assemble as higher order multiple OB domain-containing oligomers [5–8]. The complex SSBs have multiple OB domains that can be present within different subunits of the RPA complex. In humans, there are four described SSBs:, the simple SSBs, which include mitochondrial SSB (mtSSB), hSSB1 and hSSB2, and the more complex RPA, which is a heterotrimeric complex composed of RPA70, RPA32 and RPA14 [5]. The mtSSBis encoded within the nuclear genome and functions in DNA repair and replication within mitochondria. RPA is the best described SSB family member in humans and was believed until 2008 to be the only functional nuclear representative of this family in humans. However, in 2008 our group was the first to describe that humans had two further simple SSBs, which we named hSSB1 and hSSB2 [8]. While hSSB2 appears to be expressed in a few specific tissues, the exact role of this SSB is not yet clear. However, we and others have started to unravel the importance of hSSB1 in genomic stability, in cell cycle regulation and in transcription. In the current review, we summarise the multiple cellular roles hSSB1 plays to preserve genomic stability (shown in Fig. 1) as well as the underlying molecular and structural details of these processes.

There are many forms of DNA damage that can occur within the cell, however, the introduction of double strand DNA breaks (DSBs) is the most critical for a cell. Failure to repair just one DSB can result in chromosomal fragmentation during mitosis and cell death. There are two major mechanisms deployed by a cell to repair such lesions; non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ represents a less complex form of repair where the two broken ends are joined together through ligation, however, if the ends do not match (where one or both have a noncompatible ssDNA overhang) processing must occur and therefore genetic information can be lost during this process. In contrast, HR utilises the sister chromatid in order to copy and replace any lost genetic code and thus is a high-fidelity process. This can only occur when the sister is present in late S-phase and the G2 phase of the cell cycle. NHEJ is active within all phases of the cell cycle, however, in S-phase NHEJ is suppressed at stalled and collapsed DNA replication forks in order to stop the potential translocation of genetic material between chromosomes. To date, hSSB1 has only been observed to function in homologous recombination. However, as hSSB1 forms distinct foci in all phases of the cell cycle, following induction of DSBs by ionizing radiation, it is likely that it may also have a yet undescribed role to play in NHEJ. After the formation of DSBs, the cell responds rapidly through the ATM and ATR DNA repair kinases. These kinases initiate a signalling cascade that modifies and recruits repair proteins, including nucleases that are required to process the DNA ends in a 5 -3 direction. Further, these kinases initiate the cellular checkpoints that are necessary to ensure that chromosomal stability is maintained. The majority of cellular DSBs are generated by endogenous factors and ultimately produce ends that have ssDNA overhangs. Indeed, the induction of DSBs by ionising radiation (IR), is commonly used to study HR, due to the formation of two proximal ssDNA breaks on opposing strands of the DNA helix. The duplex DNA between these breaks then separates forming a “sticky” ended DNA break [9]. The ATM kinase appears to respond better to these sticky breaks as opposed to true blunt-ended breaks. One critical function of the ATM and ATR kinases is the signalling to the cell cycle checkpoints activation (e.g., CHK1, CHK2, p53). This initiates an arrest of the cell cycle that is only released once the repair process is complete. The ATM kinase also targets DNA repair proteins such as NBS1, Mre11, EXO1 and the histone variant H2 AX [10–13]. The ATM signal itself however, is dependent on the prior recruitment of the MRN (Mre11, Rad50, NBS1) complex to the break site [11,12,14,15]. Further the MRN-dependent processing of the DSB

Fig. 1. Summary of roles that hSSB1 plays in various important DNA transactions. While recent studies by us and others have shown functional involvement of hSSB1 at stalled replication folks (left in Figure, chapter 3) as well as the repair of oxidative DNA damage and double-stranded breaks (right in Figure, chapters 2 and 4), the role in mismatch repair has not been described yet. hSSB1 has also been implicated in the regulation of transcription and the cell cycle (bottom of Figure, chapters 5 and 6).

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induces the recruitment of MDC1 and the subsequent activation and maintenance of the ATM kinase activity [16]. The MRN complex contains a flexible MRE11 dimer enfolded by a single NBS1 protein, with two RAD50 subunits that function to hold together both ends of the DNA break [17–20]. The complex appeared to be the first repair component to localise to the DSB site, however, in 2008 our group demonstrated that hSSB1 was in complex with MRN and was required for MRN recruitment to the break site [8]. While MRN requires hSSB1 to be recruited to DSBs, hSSB1 does not require MRN for recruitment. Consistent with the failure of MRN to recruit to breaks sites, hSSB1 depletion prevented the initiation of cellular checkpoints following DSB induction. Following IR treatment hSSB1 is phosphorylated on T117 by the ATM kinase, with this phosphorylation being required for the extension of the hSSB1 signal away from the initial foci [18,20]. This suggests that hSSB1 may have two functions in the repair of DSBs by HR. Initially, hSSB1 binds to and sequesters the ssDNA exposed at the destabilised break ends and secondly, after recruitment of MRN and the resection of the duplex DNA to expose ssDNA, hSSB1 binds to this newly exposed substrate [8,21,22]. In vitro data support the ability of hSSB1 to bind to exposed 6 nucleotide (nt) ssDNA overhangs, as well as to a 33 basepair (bp) dsDNA oligonucleotide (likely through exposure of the ssDNA during duplex breathing) [21]. Further, it is known that hSSB1 is recruited extremely rapidly to the break site; recruitment of hSSB1 to the DSBs occurs within 10 s of DNA damage in all interphase cells. In addition, hSSB1 was seen to be recruited to alpha particle induced DSBs within 3 s [21,22]. What is not yet clear is how the RPA complex and hSSB1 function together at the break site. While RPA is recruited after hSSB1, hSSB1 foci can still be observed at 4 hours post DSB induction, so hSSB1 is likely involved both at early and later stages of repair. It is possible that there is a dynamic interchange between hSSB1 and RPA during the orchestration of repair, with each functioning in very precise transactions. Interestingly, hSSB1 interacts with NBS1 via its N-terminal BRCT domains, suggesting that hSSB1 needs to be phosphorylated prior to this interaction. The ability of hSSB1 to interact with NBS1 is impaired in a mutant associated with Nijmegen Breakage Syndrome [21], this is consistent with the impaired ability of the MRN complex containing this mutant NBS1, to be recruited to DSBs, potentially shedding light on the molecular pathology underlying Nijmegen Breakage Syndrome [23]. MRN is known to have weak nuclease activity in vitro, suggesting that additional factors are required to stimulate processing. In vitro data demonstrated that the MRN nuclease activity is substantially stimulated in the presence of hSSB1 [21] indicating that the presence of hSSB1 may facilitate faster recruitment, and thus processing, by MRN. It is still not entirely clear if hSSB1 is the first repair component to be recruited to the DSB within the cell. Indeed, Zhang et al., [24] demonstrated that the OB fold of hSSB1 also binds to poly(ADPribose) and this function is required for hSSB1 recruitment to the break site [24]. What is not known, however, is if this binding is required for the initial recruitment of hSSB1 or for the more easily observed extension of hSSB1 away from the break site. While hSSB1 interacts directly with the MRN complex, both hSSB1 and 2 have also been demonstrated to be separate components of the sensor of single-stranded DNA 1 and 2 (SOSS1 and 2) complexes, along with the integrator complex subunit 3 (INTS3) and hSSB-interacting protein 1 (hSSBIP1) [25–27]. The role of this complex is not yet clear, however, it is observed to localise to a proportion of DSB sites induced by IR [27] and is also associated with stalled and collapsed DNA replication forks [28], it has also been shown to play a critical role in transcription [29,30]. In HR, the SOSS1 components have been demonstrated to promote exonuclease 1 (EXO1) DSB resection in vitro [31]. Interestingly, while the SOSS1 complex can bind ssDNA, it does so with much reduced kinetics as compared to hSSB1 alone. This may suggest that ssDNA

3

is not the intended substrate of SOSS1 and that finding a true substrate could shed light on the function of this protein complex. Further clouding the role of the SOSS1 complex, a study by Skarr et al. demonstrated that depletion of IntS3 (part of the SOSS1 complex) by siRNA, resulted in a loss of hSSB1 expression within the cell and that the observed HR defect observed in IntS3 depleted cells could be rescued by ectopically expressing hSSB1 from a CMV promoter [27]. The role of hSSB1 in transcription will be discussed in greater detail later in this review. Rad51 is recruited to DSBs sites by BRCA2 immediately prior to invasion of the sister chromatid [26]. hSSB1 and RAD51 were observed to co-localise at break sites around 4 hours post IR treatment. Further, Rad51 and hSSB1 were seen to coimmunoprecipitate from cell lysates and to interact directly through in vitro pulldowns of recombinant hSSB1 and Rad51 [8]. Further, hSSB1 was observed to stimulate Rad51 strand invasion in a D-loop assay [8] suggesting that Rad51 and hSSB1 could be functioning together to facilitate strand invasion. This ability to interact with and stimulate the activity of Rad51 suggests that hSSB1 may share overlapping functionality with RPA. Interestingly however, RPA and hSSB1 are not shown to directly co-localise when using high resolution microscopy. Instead the foci are proximal, again supporting the very dynamic nature of hSSB1-RPA exchange during repair and that both proteins likely have precise substrates and roles to play [5].

3. The role of hSSB1 in the repair of stalled and collapsed replication forks When a replication fork is damaged or stalled, it is essential for the maintenance of cellular integrity that the retention of replication factors occurs, enabling the replication fork to efficiently restart following fork repair or lesion removal (reviewed in [32,33]). Several structures can stall DNA replication forks including transcription complexes, secondary DNA structures and a number of DNA lesions including inter-strand crosslinks and chemically modified bases. Blockages of DNA polymerase progression can lead to uncoupling of the replicative polymerase and helicase activities. This leads to long stretches of single-stranded DNA created either by the helicase continuing or by the action of nucleases such as Mre11 [34–36]. Recently, a role has been defined for hSSB1 in the stabilisation and restart of stalled and collapsed replication forks [28]. Unlike the other prominent mammalian SSB protein, the RPA heterotrimer, which is found at undamaged replication forks, hSSB1 associates with stalled/collapsed replication forks. In contrast to RPA, hSSB1 cannot be detected at replication forks during unperturbed Sphase, suggesting a specific role for hSSB1 in the repair of damaged replication forks. hSSB1 is recruited to chromatin and colocalises with other proteins required for replication fork stabilisation and repair following treatment of cells with agents such as hydroxyurea (HU) and camptothecin that stall replication forks. Once replication fork progression is impeded and stalling occurs, the ATR kinase is activated and subsequently activates other repair proteins via phosphorylation, leading to cell cycle checkpoint activation and initiation of DNA repair (reviewed in [37]). This then stimulates the recruitment of other proteins, including the MRN complex and RPA32 to aid in the repair process. Supporting a role of hSSB1 in this process, the recruitment of ATR, the MRN complex and RPA32 were shown to be dependent on hSSB1 following replication fork collapse [28]. Upon replication fork stalling the checkpoint kinase Chk1 is phosphorylated and activated by ATR [38]. Checkpoint signalling functions to down-regulate origin firing and stimulates proteins involved in fork stabilisation. Following replication fork stalling and collapse, hSSB1 was found to be required for the phosphorylation of

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the Chk1 checkpoint kinase on several sites, suggesting that hSSB1 is required for checkpoint activation [28]. A number of mechanisms can be utilised to resolve a stalled replication fork including removal of the lesion/barrier, or by lesion bypass, through additional pathways including HR (reviewed in [36]). Stalled forks may regress into 4-way Holliday Junction structures to aid the repair process. Although the Rad51 recombinase is most well known as a protein involved in HR processes, it has been shown that stalled forks can be restarted via a Rad51-dependent pathway, independently of a homologous template and may therefore be distinct from classical HR [39]. If replication forks are stalled for a prolonged period, forks may collapse and can be processed into DSBs, which are repaired by the classical HR pathway [39–43]. Following cellular hSSB1 depletion and HU treatment, more DNA double-strand breaks could be detected, suggesting that more collapsed replication forks were present in the absence of hSSB1 and that it is required for replication fork stability. Using a DNA fibre assay, hSSB1 was also shown to be required for overall replication restart, following fork stalling by HU [28]. A recent paper also addressed the regulation of hSSB1 following replication fork stalling [44]. The kinase DNA PK was observed to phosphorylate hSSB1 on Serine 134 (S134) following replication fork stress. This protein modification was also found to be regulated by PPP-family phosphatases that function to continually remove the phosphate group in undamaged cells. The continual removal of the phosphate groups from this site may serve to keep the system primed in order to initiate a rapid response upon replication inhibition. It was suggested that this modification was unlikely to alter the DNA binding ability of hSSB1, but the specific function of this phosphorylation event is unknown. However, expression of a non-phosphorylatable S134A mutant was unable to rescue the cellular sensitivity of hSSB1-depleted cells to replication fork stalling agents, suggesting that this modification is required for the function of hSSB1 in the recovery of stalled replication forks [44]. Supporting the data from human cells, mouse studies indicate that although the murine homologue of hSSB1 (mSsb1) is essential for genome maintenance, this is not via activation of cell cycle checkpoints or DNA double-strand break recognition. In contrast, mSsb1 is suggested to specifically function in the repair of replication-associated DNA damage. In addition, the mouse Ssb1 and Ssb2 seem to compensate for the loss of either protein. Loss of mouse Ssb1 results in upregulation of mSsb2 and vice versa. Interestingly, the depletion of both Ssb1 and 2 in mice leads to a replication-stress induced severe genome instability phenotype, with conditional knockout leading to embryonic lethality by embryonic day 10.5 [45]. Further investigation is required to completely characterise the role of mSsb1 in this process [46,47].

4. The role of hSSB1 in the base excision repair (BER) pathway The mammalian genome is under constant insult from environmental and physiological conditions (e.g.,endogenous aerobic metabolism, exogenous chemicals, sunlight, reactive oxygen species - ROS) resulting in a variety of modifications to DNA bases [48]. Similar to the aforementioned repair of stalled or collapsed replication forks and DSBs, cells have sophisticated mechanisms to detect and remove these DNA lesions. The two described mechanisms to repair these damaged bases in mammals are the base excision repair (BER) and nucleotide excision repair (NER) pathways. Whether hSSB1 functions in the NER pathway remains unknown but this protein has recently been implicated in the BER pathway, specifically in the repair of oxidised DNA bases [49,50]. The BER pathway is an integral repair mechanism with loss of this system during development yielding embryonic lethality in

animal models [51–53]. This pathway is the predominant mechanism for the repair of oxidised, deaminated or alkylated DNA bases. These lesions tend not to distort the DNA helix or impede the transcription process. However, if such damaged DNA bases are left to accumulate, as is the case in the failure to repair oxidised guanine bases, transcriptional mutagenesis will result [54]. Such mutagenesis signatures are often observed in cancers [55,56]. Classically, removal of the damaged base commences with a specific mono- or bi-functional DNA glycosylase that either hydrolyses the N-glycosylic bond of a damaged base or completely cleaves the DNA. At least 11 mammalian glycosylases exist to specifically remove the various types of damaged bases, making BER a versatile pathway [57]. Cleavage by the glycosylase creates an apurinic or apyrimidinic site (AP site) which is further processed by the endonuclease APE1 to generate an ssDNA break [58–60]. Using the undamaged strand as a template, DNA polymerase ␤ then replaces the removed single nucleotide base, in the case of short-patch-BER [61]. While DNA polymerase ␤ also functions in long-patch-BER, which is the repair of two or more damaged bases, this polymerase only incorporates the first nucleotide with replicative polymerases required to continue elongation of the nascent strand [62]. DNA ligase III in complex with XRCC1 is then recruited to ligate the nicked strand and complete the BER process. Experimental evidence positions hSSB1 as an early participant in the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) by BER. ROS and cellular oxidative damage modify guanine bases to generate the ubiquitous 8-oxoG DNA lesion. The bi-functional human 8oxoguanine glycosylase 1 (hOGG1) is responsible for the removal of 8-oxoG lesions opposite cytosine bases but not opposite adenosine bases [59,63–65]. Using in vitro techniques, hSSB1 was found to bind dsDNA containing 8-oxoG (but not undamaged dsDNA) and form a protein complex with hOGG1 [50].These in vitro analyses also indicated that hSSB1 has the ability to recruit and enhance hOGG1 cleavage activity at damaged guanine bases. Consistent with these in vitro data, cells depleted of endogenous hSSB1 were sensitive to oxidative stress-inducing agents (H2 O2 and potassium bromate) and exhibited a failure to efficiently reduce nuclear 8oxoG levels. In keeping with these cellular observations, reduced hOGG1 recruitment to detergent-resistant chromatin-like structures was observed in oxidative stress treated cells depleted of hSSB1. Whether hSSB1 also functions during the later stages of BER while abetting the recognition and specific glycosylase-mediated removal of other DNA base modifications remains to be determined. More recent studies have addressed the molecular regulation of hSSB1 whilst functioning in the BER pathway. In response to cellular oxidative stress, hSSB1 has been shown to self-oligomerise into dimers and tetramers, which are essential for efficient BER [49,66]. Interestingly, while oligomer formation is necessary for hSSB1 to participate in BER, oligomer formation is not likely to prevent hSSB1 from binding other SOSS1 complex components (see also Section 7 below).

5. Regulation of hSSB1 by INTS3 and their role in transcription termination In addition to participating in multiple DNA repair pathways, hSSB1 is also known to play a role in transcription, as part of the Integrator complex, and interestingly hSSB1 is also regulated by this complex. Integrator is a 12-subunit complex that was initially demonstrated to participate in the 3 processing of small nuclear RNAs (snRNAs) [67]. Several studies have now shown that a proportion of cellular hSSB1 exists in complex with the Integrator subunit 3 (INTS3) [25–27,68] and the low molecular weight protein hSSBIP1/LOC58493/c9orf80 [25–27] (SOSS1 complex, see

Please cite this article in press as: L.V. Croft, et al., Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.03.014

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also Introduction). The hSSB1-INTS3 interaction is mediated by the OB domain of hSSB1 [27,68] and the N-terminal region containing the DUF2356 domain of INTS3 [25,27,69]. Although the INTS3-hSSB1 interaction occurs constitutively and independently of DNA damage [27], INTS3 is required for the activation of the hSSB1-dependent DNA damage response [25–27]. Interestingly, this dependence on INTS3 is due at least in part to the regulation of hSSB1 mRNA expression as siRNA-mediated depletion of INTS3 leads to an almost complete loss of hSSB1 mRNA and consequently protein [27]. These studies thus suggest that INTS3 plays a role in regulating hSSB1 function in the DNA damage response by regulating cellular levels of hSSB1 at the transcriptional level. More recent studies suggest that hSSB1 and INTS3 also function together with other components of the Integrator complex and RNA Polymerase II in transcription termination [29]. Using High Throughput Sequencing (HIT-Seq) analysis Skaar et al., demonstrated that INTS3 and hSSB1 associate with the Integrator complex at several sites in the genome including: small nuclear RNA (snRNA) loci, 3 end of replication-dependent histone genes and at promoter proximal sites in a subset of genes with polyadenylated mRNAs [29]. Using chromatin immunoprecipitation (ChIP), hSSB1 along with INTS3 were detected at the U2 snRNA locus, at the replication dependent histone gene Histone H2 A locus, and at the promoter proximal site of the polyadenylated transcript encoding JunB [29]. Despite its presence at these sites, siRNA-mediated depletion of hSSB1 only led to a modest effect on processing of snRNA, replication-dependent histone genes and polyadenylated mRNAs, while INTS3 depletion had a more profound effect on processing of these genes. Moreover, the INTS3-hSSB1 complex was shown to interact with the transcription termination factors Negative Elongation Factor (NELF) and the DRB Sensitivity-Inducing Factor (DSIF). The NELF and DSIF complexes function in promoter proximal pausing of the C-terminal domain of RNA polymerase II during transcription, a mechanism proposed to be a key regulatory step in transcription of highly inducible, stress response genes [29,70,71]. It is suggested that the INTS3-hSSB1 complex may be involved in binding the ssDNA in the transcription bubble at RNA Pol II pause sites, and together with the helicase function of the Integrator INTS6 subunit, it may work to resolve RNA-DNA hybrids in transcription bubbles [29]. These studies thus demonstrate that in addition to its function in multiple DNA repair pathways, hSSB1 also plays a role in transcription termination as part of the Integrator complex.

6. hSSB1 and cell cycle regulation Eukaryotic cell division requires a high level of regulation in order to maintain genomic stability. Cell cycle checkpoints are key mechanisms that ensure accurate propagation of DNA during cell division. Our group and others have shown that hSSB1 is also involved in cell cycle checkpoint activation [8,30,72]. Our initial studies showed that cells depleted of hSSB1 exhibit defective G1 and G2 cell cycle checkpoint activation in response to ionising radiation-induced DNA damage [8]. Indeed, autophosphorylation of the ATM kinase as well as ATM-mediated phosphorylation of downstream checkpoint proteins such as p53, Chk2, Chk1 and NBS1 are defective in hSSB1 depleted cells, thus suggesting that hSSB1 also plays a central role in DNA damage-induced cell cycle checkpoint activation. Other studies have demonstrated that hSSB1 regulates the G1/S transition during the cell cycle and the damageinduced G2/M checkpoint by also interacting with p21 and p53, both key regulators of the cell cycle [30,72]. hSSB1 binds to p21 and p53 and prevents their ubiquitin-mediated degradation, thus positively regulating their stability. hSSB1 depletion leads to de-stabilisation of p21, which induces enhanced G1/S transition

5

and abrogates IR-induced G2/M checkpoint activation [72]. hSSB1 depletion also leads to de-stabilisation of p53, causing a defective p53-dependent G2/M checkpoint activation in response to DNA damage. Moreover, hSSB1 also interacts with the acetyltransferase p300 and enables p53 acetylation at Lys382, an event that regulates the transcriptional activity of p53 and consequently the p53-induced expression of p21 [30]. These studies thus demonstrate that hSSB1 regulates cell cycle transition in unperturbed cells, but also plays an important role in DNA damage-induced cell cycle check point activation via both the ATM and p53 pathways.

7. Structural aspects of hSSB1 The OB fold, which is the defining part of hSSB1, was originally characterised from a subset of bacterial and yeast proteins as a folding motif that binds oligonucleotides or oligosaccharides [73]. Studies have since determined that OB domains also have the ability to form protein-DNA, protein-RNA and protein-protein interactions [74,75]. As delineated in several of the reviews in this issue, the domain organisation of the DNA binding OB domains of a number of SSBs has been well characterised. For example, the SSB from Escherichia coli (EcoSSB) is a simple SSB that oligomerises to form a functional OB homotetramer [76–78] (see the review by Antony and Lohman in this issue). In contrast, RPA, a complex SSB, exists in a heterotrimeric arrangement composed of six OB domains, from three different subunits: RPA70, RPA32 and RPA14 [79–81] (see the review by Byrne and Oakley in this issue). The SSB that is structurally most similar to hSSB1, the SSB from Sulfolobus solfataricus (SsoSSB), is a simple SSB and exists exclusively as a monomer [82]. The number and sequence of amino acids present in OB domains varies considerably between proteins, however, there are numerous structural features that are common to all OB folds. OB folds from all three domains of life contain five antiparallel ␤-strands, coiled to form a barrel structure which is capped by an ␣-helix (between the third and fourth ␤-strands) on one end, and contains a DNA binding cleft on the other [73]. The hSSB1 monomer (<20% sequence similarity to other SSBs) contains a single, highly conserved OB fold at the N-terminus, followed by a flexible, spacer region and a conserved C-terminal tail. The structure of full length hSSB1 (1–211) was first solved in complex with INTS3 as a part of the SOSS1 complex [83] (see also sections above). The published X-ray crystal structure of this complex (PDB ID 40WX) demonstrated that residues 5–109 of hSSB1 form a typical OB fold composed of five anti-parallel ␤-strands (␤1, ␤3, ␤4, ␤5 and ␤6), capped by an ␣-helix (␣1) between ␤3 and ␤4. Within the SOSS1 complex, the hSSB1 protein is the sole component that is responsible for the interaction with ssDNA. While hSSB1 interacts with SOSSA in the crystal structure via strands ␤1, ␤6, and ␣1, the ␣1 – ␤5 loop and the C-terminal tail, the hSSB1-ssDNA interaction is facilitated by residues 2–16 which form a DNA binding groove at the N-terminus, as well as loops ␤2-␤3 and ␤4-␣1 and strands ␤4, ␤5, and ␤6 [83]. DNA binding is mediated by electrostatic interaction, hydrogen bonding and base-stacking involving residues T32, K33, W55, D56, Y74, F78, Y85 and R88. Interestingly, the solution structure of the OB domain of hSSB1 bound to ssDNA determined by our group (shown in Fig. 2A) revealed significant differences to the published crystal structure [83–85]. Kariawasam et al., carried out chemical shift mapping of the ssDNA binding interface of hSSB1 by NMR in solution and uncovered a set of residues that exhibit substantial shifts but are not involved in binding DNA in the crystal lattice. This might be due to non-native crystal contacts between SOSSA and the ssDNA that caused distortion of the ssDNA, as discussed by the authors of all three studies [83–85].

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Fig. 2. A. NMR-based structural model of hSSB1 bound to ssDNA. The aromatic residues (W55, Y74, F78 and Y85) that intercalate with the ssDNA are shown as green sticks. B. Model of oxidative hSSB1 tetramer based on NMR and mutagenesis. The intermolecular disulphide bonds (residues C81 and C99) are labelled. The hSSB1 monomers are shown in blue, dark grey, green and salmon, respectively.

Further structural analysis by NMR as well as biophysical and functional experiments of DNA binding by hSSB1 revealed additional important differences between the crystal structure and the solution structure [85]. Perhaps the most important difference is the number of aromatic resides of hSSB1 that were found to intercalate with the ssDNA bases. In the solution structure, recognition of ssDNA is modulated by four aromatics (W55, Y74, F78 and Y85; Fig. 2A), similar to the structurally homologous SsoSSB [86], whereas in the crystal structure only two were determined to be involved (W55 and F78). Further, the spacing between all four aromatics with respect to the ssDNA is different to the one found by Ren et al. [83] and substantial differences in the structural conformation of the ssDNA also exist. While hSSB1 is a monomer under non-oxidising conditions, as we have detailed in Section 4, hSSB1 plays a crucial role as a functional oligomer in the repair of DNA under oxidative conditions. hSSB1 is required for the removal of 8-oxoG from the genome through BER, where oligomeric hSSB1 localises to sites of damage and recruits hOGG1 [50]. Notably, in contrast to oligomeric hSSB1, monomeric hSSB1 has been found to display a significantly lower affinity for oxidised DNA which results in an 8-oxoG repair defect as well as the absence of ATM signalling [49]. There are three cysteine residues present in the OB fold of hSSB1, two of which are solvent exposed (C81 and C99) [49]. These cysteine residues are likely to facilitate the oligomerisation of hSSB1 under oxidative conditions through the formation of disulphide bonds [85]. In addition to C81 and C99, NMR experiments carried out under oxidising conditions also revealed a set of charged and hydrophobic residues (N16, N18, K33, T71, K72, R88 and D91) within the OB domain of hSSB1 that are also involved in oligomer formation [66]. All of these residues, along with C81 and C99, were found to be located on two distinct sides of the hSSB1 protein. Informed by mutational data and the tetrameric structure of EcoSSB (PDB 1SRU) [76,78], a molecular model of oligomeric hSSB1 has been calculated [66] (shown in Fig. 2B). This model suggests that hSSB1 can exist as a functional tetramer, with monomer-monomer and dimer-dimer interactions occurring at distinct surfaces of the OB domain. Importantly, as implied by the structure of the tetramer, hSSB1 oligomerisation does not preclude interaction with both the SOSS1 and MRN complex, respectively. In addition, the interface for the hSSB1-hSSB1 interaction does not overlap with the ssDNA binding surface. However, the molecular details of how hSSB1 dimers or tetramers recognise DNA as well as the differences in the binding affinity between monomeric and oligomeric hSSB1 [50] are yet to be determined.

8. Concluding remarks Since its discovery in 2008, it is becoming increasingly evident that the hSSB1 protein is essential for the maintenance of genome stability. It is involved in DNA transactions that repair DNA damage at chromatin (such as in the HR or BER pathways) as well as at stalled replication forks. Moreover, it has important roles in the regulation of transcription termination and the cell cycle (summarised in Fig. 1). While this review has focused on the known roles of hSSB1 in DNA repair, this important protein is most likely also involved in other cellular DNA transactions, awaiting further elucidation in the near future. Classical chemo- or radiotherapy cancer treatments aim at efficiently killing cancer cells by inducing DNA damage and driving apoptosis. However, cancer cells display a high rate of DNA repair that effectively counteract these classical therapies, ensuring cancer cell survival. Innovative solutions to this problem lie in therapies to specifically inhibit DNA repair activities, enhancing the effects of current DNA-damaging anti-cancer treatments. Examples where this approach has been successfully exploited include the development of Poly (ADP-ribose) polymerase (PARP) family protein inhibitors in BRCA-associated breast and ovarian cancer [87,88] and the inhibition of BRCA1 (breast cancer protein 1) in sporadic ovarian cancer [89]. Importantly, structural analysis, together with functional data has allowed us to characterise hSSB1-DNA interactions at a molecular level. This detailed mechanistic understanding of the structure and function of hSSB1 in DNA repair will make the future design of tailored hSSB1 inhibitors as potentially less toxic anti-cancer treatments a real possibility.

Acknowledgements This work was supported by the Chenhall Research Trust Fellowship to DJR, by an Advance Queensland Research Fellowship to EB and by an NHMRC Early Career Fellowship to MNA (1091589).

References [1] S. Broderick, K. Rehmet, C. Concannon, H.P. Nasheuer, Eukaryotic single-stranded DNA binding proteins: central factors in genome stability, Subcell. Biochem 50 (2010) 143–163. [2] R.L. Flynn, L. Zou, Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians, Crit. Rev. Biochem Mol. Biol. 45 (4) (2010) 266–275. [3] M.P. Horvath, Structural anatomy of telomere OB proteins, Crit. Rev. Biochem. Mol. Biol. 46 (5) (2011) 409–435.

Please cite this article in press as: L.V. Croft, et al., Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.03.014

G Model YSCDB-2550; No. of Pages 8

ARTICLE IN PRESS L.V. Croft et al. / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

[4] D.J. Richard, E. Bolderson, K.K. Khanna, Multiple human single-stranded DNA binding proteins function in genome maintenance: structural, biochemical and functional analysis, Crit. Rev. Biochem. Mol. Biol. 44 (2-3) (2009) 98–116. [5] N.W. Ashton, E. Bolderson, L. Cubeddu, K.J. O’Byrne, D.J. Richard, Human single-stranded DNA binding proteins are essential for maintaining genomic stability, BMC Mol. Biol. 14 (2013) 9. [6] P.K. Datta, H.L. Moses, STRAP and Smad7 synergize in the inhibition of transforming growth factor beta signaling, Mol. Cell. Biol. 20 (9) (2000) 3157–3167. [7] M.T. Oliveira, L.S. Kaguni, Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein, PLoS One 5 (10) (2010) e15379. [8] D.J. Richard, E. Bolderson, L. Cubeddu, R.I. Wadsworth, K. Savage, G.G. Sharma, M.L. Nicolette, S. Tsvetanov, M.J. McIlwraith, R.K. Pandita, S. Takeda, R.T. Hay, J. Gautier, S.C. West, T.T. Paull, T.K. Pandita, M.F. White, K.K. Khanna, Single-stranded DNA-binding protein hSSB1 is critical for genomic stability, Nature 453 (7195) (2008) 677–681. [9] B. Shiotani, L. Zou, Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks, Mol. Cell. 33 (5) (2009) 547–558. [10] E. Bolderson, N. Tomimatsu, D.J. Richard, D. Boucher, R. Kumar, T.K. Pandita, S. Burma, K.K. Khanna, Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks, Nucleic Acids Res. 38 (6) (2010) 1821–1831. [11] A.W. Kijas, Y.C. Lim, E. Bolderson, K. Cerosaletti, M. Gatei, B. Jakob, F. Tobias, G. Taucher-Scholz, N. Gueven, G. Oakley, P. Concannon, E. Wolvetang, K.K. Khanna, L. Wiesmuller, M.F. Lavin, ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1, Nucleic Acids Res. 43 (17) (2015) 8352–8367. [12] J.H. Lee, T.T. Paull, ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex, Science 308 (5721) (2005) 551–554. [13] T. Uziel, Y. Lerenthal, L. Moyal, Y. Andegeko, L. Mittelman, Y. Shiloh, Requirement of the MRN complex for ATM activation by DNA damage, EMBO J. 22 (20) (2003) 5612–5621. [14] C.T. Carson, R.A. Schwartz, T.H. Stracker, C.E. Lilley, D.V. Lee, M.D. Weitzman, The Mre11 complex is required for ATM activation and the G2/M checkpoint, EMBO J. 22 (24) (2003) 6610–6620. [15] J. Falck, J. Coates, S.P. Jackson, Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage, Nature 434 (7033) (2005) 605–611. [16] G.S. Stewart, B. Wang, C.R. Bignell, A.M. Taylor, S.J. Elledge, MDC1 is a mediator of the mammalian DNA damage checkpoint, Nature 421 (6926) (2003) 961–966. [17] K.P. Hopfner, A. Karcher, L. Craig, T.T. Woo, J.P. Carney, J.A. Tainer, Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase, Cell 105 (4) (2001) 473–485. [18] K. Lammens, D.J. Bemeleit, C. Mockel, E. Clausing, A. Schele, S. Hartung, C.B. Schiller, M. Lucas, C. Angermuller, J. Soding, K. Strasser, K.P. Hopfner, The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair, Cell 145 (1) (2011) 54–66. [19] Y.B. Park, J. Chae, Y.C. Kim, Y. Cho, Crystal structure of human Mre11: understanding tumorigenic mutations, Structure 19 (11) (2011) 1591–1602. [20] C.B. Schiller, K. Lammens, I. Guerini, B. Coordes, H. Feldmann, F. Schlauderer, C. Mockel, A. Schele, K. Strasser, S.P. Jackson, K.P. Hopfner, Structure of Mre11-Nbs1 complex yields insights into ataxia-telangiectasia-like disease mutations and DNA damage signaling, Nat. Struct. Mol. Biol. 19 (7) (2012) 693–700. [21] D.J. Richard, L. Cubeddu, A.J. Urquhart, A. Bain, E. Bolderson, D. Menon, M.F. White, K.K. Khanna, hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity, Nucleic Acids Res. 39 (9) (2011) 3643–3651. [22] D.J. Richard, K. Savage, E. Bolderson, L. Cubeddu, S. So, M. Ghita, D.J. Chen, M.F. White, K. Richard, K.M. Prise, G. Schettino, K.K. Khanna, hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex, Nucleic Acids Res. 39 (5) (2011) 1692–1702. [23] H. Tauchi, S. Matsuura, J. Kobayashi, S. Sakamoto, K. Komatsu, Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability, Oncogene 21 (58) (2002) 8967–8980. [24] F. Zhang, Y. Chen, M. Li, X. Yu, The oligonucleotide/oligosaccharide-binding fold motif is a poly(ADP-ribose)-binding domain that mediates DNA damage response, Proc. Natl. Acad. Sci. U. S. A. 111 (20) (2014) 7278–7283. [25] J. Huang, Z. Gong, G. Ghosal, J. Chen, SOSS complexes participate in the maintenance of genomic stability, Mol. Cell. 35 (3) (2009) 384–393. [26] Y. Li, E. Bolderson, R. Kumar, P.A. Muniandy, Y. Xue, D.J. Richard, M. Seidman, T.K. Pandita, K.K. Khanna, W. Wang, HSSB1 and hSSB2 form similar multiprotein complexes that participate in DNA damage response, J. Biol Chem. 284 (35) (2009) 23525–23531. [27] J.R. Skaar, D.J. Richard, A. Saraf, A. Toschi, E. Bolderson, L. Florens, M.P. Washburn, K.K. Khanna, M. Pagano, INTS3 controls the hSSB1-mediated DNA damage response, J. Cell. Biol. 187 (1) (2009) 25–32. [28] E. Bolderson, E. Petermann, L. Croft, A. Suraweera, R.K. Pandita, T.K. Pandita, T. Helleday, K.K. Khanna, D.J. Richard, Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks, Nucleic Acids Res. 42 (10) (2014) 6326–6336. [29] J.R. Skaar, A.L. Ferris, X. Wu, A. Saraf, K.K. Khanna, L. Florens, M.P. Washburn, S.H. Hughes, M. Pagano, The integrator complex controls the termination of

[30]

[31]

[32] [33] [34]

[35]

[36] [37] [38]

[39]

[40] [41] [42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51]

[52] [53]

[54]

[55]

7

transcription at diverse classes of gene targets, Cell. Res. 25 (3) (2015) 288–305. S. Xu, Y. Wu, Q. Chen, J. Cao, K. Hu, J. Tang, Y. Sang, F. Lai, L. Wang, R. Zhang, S.P. Li, Y.X. Zeng, Y. Yin, T. Kang, hSSB1 regulates both the stability and the transcriptional activity of p53, Cell. Res. 23 (3) (2013) 423–435. S.H. Yang, R. Zhou, J. Campbell, J. Chen, T. Ha, T.T. Paull, The SOSS1 single-stranded DNA binding complex promotes DNA end resection in concert with Exo1, EMBO J. 32 (1) (2013) 126–139. D. Cortez, Preventing replication fork collapse to maintain genome integrity, DNA Repair. (Amst) 32 (2015) 149–157. M. Berti, A. Vindigni, Replication stress: getting back on track, Nat. Struct. Mol. Biol. 23 (2) (2016) 103–109. J. Walter, J. Newport, Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha, Mol. Cell 5 (4) (2000) 617–627. A.M. Leon-Ortiz, J. Svendsen, S.J. Boulton, Metabolism of DNA secondary structures at the eukaryotic replication fork, DNA Repair. (Amst) 19 (2014) 152–162. E. Petermann, T. Helleday, Pathways of mammalian replication fork restart, Nat. Rev. Mol. Cell. Biol. 11 (10) (2010) 683–687. S.A. Yazinski, L. Zou, Functions, Regulation, and Therapeutic Implications of the ATR Checkpoint Pathway, Annu Rev. Genet. 50 (2016) 155–173. Z. Guo, A. Kumagai, S.X. Wang, W.G. Dunphy, Requirement for atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in xenopus egg extracts, Genes Dev. 14 (21) (2000) 2745–2756. E. Petermann, M.L. Orta, N. Issaeva, N. Schultz, T. Helleday, Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair, Mol. Cell. 37 (4) (2010) 492–502. A.M. Carr, DNA structure dependent checkpoints as regulators of DNA repair, DNA Repair. (Amst) 1 (12) (2002) 983–994. R.S. Cha, N. Kleckner, ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones, Science 297 (5581) (2002) 602–606. J.A. Cobb, T. Schleker, V. Rojas, L. Bjergbaek, J.A. Tercero, S.M. Gasser, Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations, Genes Dev. 19 (24) (2005) 3055–3069. J.M. Sogo, M. Lopes, M. Foiani, Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects, Science 297 (5581) (2002) 599–602. N.W. Ashton, N. Paquet, S.L. Shirran, E. Bolderson, R. Kariawasam, C. Touma, A. Fallahbaghery, R. Gamsjaeger, L. Cubeddu, C. Botting, P.M. Pollock, K.J. O’Byrne, D.J. Richard, hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases, DNA Repair. (Amst) 54 (2017) 30–39. W. Shi, T. Vu, D. Boucher, A. Biernacka, J. Nde, R.K. Pandita, J. Straube, G.M. Boyle, F. Al-Ejeh, P. Nag, J. Jeffery, J.L. Harris, A.L. Bain, M. Grzelak, M. Skrzypczak, A. Mitra, N. Dojer, N. Crosetto, N. Cloonan, O.J. Becherel, J. Finnie, J.R. Skaar, C.R. Walkley, T.K. Pandita, M. Rowicka, K. Ginalski, S.W. Lane, K.K. Khanna, Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress, Blood 129 (18) (2017) 2479–2492. W. Shi, A.L. Bain, B. Schwer, F. Al-Ejeh, C. Smith, L. Wong, H. Chai, M.S. Miranda, U. Ho, M. Kawaguchi, Y. Miura, J.W. Finnie, M. Wall, J. Heierhorst, C. Wicking, K.J. Spring, F.W. Alt, K.K. Khanna, Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2, PLoS Genet 9 (2) (2013), e1003298. N. Feldhahn, E. Ferretti, D.F. Robbiani, E. Callen, S. Deroubaix, L. Selleri, A. Nussenzweig, M.C. Nussenzweig, The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo, EMBO J. 31 (20) (2012) 4045–4056. T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (6422) (1993) 709–715. N. Paquet, M.N. Adams, N.W. Ashton, C. Touma, R. Gamsjaeger, L. Cubeddu, V. Leong, S. Beard, E. Bolderson, C.H. Botting, K.J. O’Byrne, D.J. Richard, hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress, Sci. Rep. 6 (2016) 27446. N. Paquet, M.N. Adams, V. Leong, N.W. Ashton, C. Touma, R. Gamsjaeger, L. Cubeddu, S. Beard, J.T. Burgess, E. Bolderson, K.J. O’Byrne, D.J. Richard, hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway, Nucleic Acids Res. 43 (18) (2015) 8817–8829. H. Gu, J.D. Marth, P.C. Orban, H. Mossmann, K. Rajewsky, Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting, Science 265 (5168) (1994) 103–106. D.M. Wilson 3rd, L.H. Thompson, Life without DNA repair, Proc. Natl. Acad. Sci. U. S. A. 94 (24) (1997) 12754–12757. S. Xanthoudakis, R.J. Smeyne, J.D. Wallace, T. Curran, The redox/DNA repair protein, ref-1, is essential for early embryonic development in mice, Proc. Natl. Acad. Sci. U. S A. 93 (17) (1996) 8919–8923. S. Shibutani, M. Takeshita, A.P. Grollman, Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG, Nature 349 (6308) (1991) 431–434. J.H. Hoeijmakers, DNA damage, aging, and cancer, N Engl. J. Med. 361 (15) (2009) 1475–1485.

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[56] C. Greenman, P. Stephens, R. Smith, G.L. Dalgliesh, C. Hunter, G. Bignell, H. Davies, J. Teague, A. Butler, C. Stevens, S. Edkins, S. O’Meara, I. Vastrik, E.E. Schmidt, T. Avis, S. Barthorpe, G. Bhamra, G. Buck, B. Choudhury, J. Clements, J. Cole, E. Dicks, S. Forbes, K. Gray, K. Halliday, R. Harrison, K. Hills, J. Hinton, A. Jenkinson, D. Jones, A. Menzies, T. Mironenko, J. Perry, K. Raine, D. Richardson, R. Shepherd, A. Small, C. Tofts, J. Varian, T. Webb, S. West, S. Widaa, A. Yates, D.P. Cahill, D.N. Louis, P. Goldstraw, A.G. Nicholson, F. Brasseur, L. Looijenga, B.L. Weber, Y.E. Chiew, A. DeFazio, M.F. Greaves, A.R. Green, P. Campbell, E. Birney, D.F. Easton, G. Chenevix-Trench, M.H. Tan, S.K. Khoo, B.T. Teh, S.T. Yuen, S.Y. Leung, R. Wooster, P.A. Futreal, M.R. Stratton, Patterns of somatic mutation in human cancer genomes, Nature 446 (7132) (2007) 153–158. [57] H.E. Krokan, R. Standal, G. Slupphaug, DNA glycosylases in the base excision repair of DNA, Biochem J. 325 (Pt 1) (1997) 1–16. [58] B. Demple, T. Herman, D.S. Chen, Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes, Proc. Natl. Acad. Sci. U. S. A. 88 (24) (1991) 11450–11454. [59] T.A. Rosenquist, D.O. Zharkov, A.P. Grollman, Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase, Proc. Natl. Acad. Sci. U. S. A. 94 (14) (1997) 7429–7434. [60] V.S. Sidorenko, G.A. Nevinsky, D.O. Zharkov, Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease, DNA Repair. (Amst) 6 (3) (2007) 317–328. [61] A.F. Moon, M. Garcia-Diaz, V.K. Batra, W.A. Beard, K. Bebenek, T.A. Kunkel, S.H. Wilson, L.C. Pedersen, The X family portrait: structural insights into biological functions of X family polymerases, DNA Repair. (Amst) 6 (12) (2007) 1709–1725. [62] P. Fortini, E. Dogliotti, Base damage and single-strand break repair: mechanisms and functional significance of short- and long-patch repair subpathways, DNA Repair. (Amst) 6 (4) (2007) 398–409. [63] M. Bjoras, L. Luna, B. Johnsen, E. Hoff, T. Haug, T. Rognes, E. Seeberg, Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites, EMBO J. 16 (20) (1997) 6314–6322. [64] S.D. Bruner, D.P. Norman, G.L. Verdine, Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA, Nature 403 (6772) (2000) 859–866. [65] T. Roldan-Arjona, Y.F. Wei, K.C. Carter, A. Klungland, C. Anselmino, R.P. Wang, M. Augustus, T. Lindahl, Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase, Proc. Natl. Acad. Sci. U. S. A. 94 (15) (1997) 8016–8020. [66] C. Touma, M.N. Adams, N.W. Ashton, M. Mizzi, S. El-Kamand, D.J. Richard, L. Cubeddu, R. Gamsjaeger, A data-driven structural model of hSSB1 (NABP2/OBFC2B) self-oligomerization, Nucleic Acids Res. 45 (14) (2017) 8609–8620. [67] D. Baillat, M.A. Hakimi, A.M. Naar, A. Shilatifard, N. Cooch, R. Shiekhattar, Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II, Cell 123 (2) (2005) 265–276. [68] F. Zhang, J. Wu, X. Yu, Integrator3, a partner of single-stranded DNA-binding protein 1, participates in the DNA damage response, J. Biol Chem. 284 (44) (2009) 30408–30415. [69] F. Zhang, T. Ma, X. Yu, A core hSSB1–INTS complex participates in the DNA damage response, J. Cell Sci. 126 (21) (2013) 4850–4855. [70] Q. Zhou, T. Li, D.H. Price, RNA polymerase II elongation control, Annu Rev. Biochem. 81 (2012) 119–143. [71] K. Adelman, J.T. Lis, Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans, Nat. Rev. Genet. 13 (10) (2012) 720–731.

[72] S. Xu, Z. Feng, M. Zhang, Y. Wu, Y. Sang, H. Xu, X. Lv, K. Hu, J. Cao, R. Zhang, L. Chen, M. Liu, J.P. Yun, Y.X. Zeng, T. Kang, hSSB1 binds and protects p21 from ubiquitin-mediated degradation and positively correlates with p21 in human hepatocellular carcinomas, Oncogene 30 (19) (2011) 2219–2229. [73] A.G. Murzin, OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences, The EMBO J. 12 (3) (1993) 861–867. [74] V. Arcus, OB-fold domains: a snapshot of the evolution of sequence, structure and function, Curr. Opin. Struct. Biol. 12 (6) (2002) 794–801. [75] D.L. Theobald, R.M. Mitton-Fry, D.S. Wuttke, Nucleic acid recognition by ob-fold proteins, Annual Rev. Biophys. Biomol. Struct. 32 (1) (2003) 115–133. [76] S. Raghunathan, A.G. Kozlov, T.M. Lohman, G. Waksman, Structure of the DNA binding domain of E. coli SSB bound to ssDNA, Nat. Struct. Biol. 7 (2000) 648. [77] T.M. Lohman, M.E. Ferrari, Escherichia Coli single-stranded DNA-binding protein: Multiple DNA-binding modes and cooperativities, Annu. Rev. Biochem. 63 (1) (1994) 527–570. [78] S. Raghunathan, C.S. Ricard, T.M. Lohman, G. Waksman, Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-Å resolution, Proc. Natl. Acad. Sci. 94 (13) (1997) 6652–6657. [79] C. Iftode, Y. Daniely, J.A. Borowiec, Replication protein A (RPA): the eukaryotic SSB, Crit. Rev. Biochem. Mol. Biol 34 (3) (1999) 141–180. [80] Y. Zou, Y. Liu, X. Wu, S.M. Shell, Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses, J. Cell. Physiol 208 (2) (2006) 267–273. [81] M.S. Wold, Replication protein A: A heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism, Annu. Rev. Biochem. 66 (1) (1997) 61–92. [82] R.I.M. Wadsworth, M.F. White, Identification and properties of the crenarchaeal single-stranded DNA binding protein from sulfolobus solfataricus, Nucleic Acids Res. 29 (4) (2001) 914–920. [83] W. Ren, H. Chen, Q. Sun, X. Tang, S.C. Lim, J. Huang, H. Song, Structural basis of SOSS1 complex assembly and recognition of ssDNA, Cell. Rep. 6 (6) (2014) 982–991. [84] R. Kariawasam, C. Touma, L. Cubeddu, R. Gamsjaeger, Backbone (1)H, (13)C and (15)N resonance assignments of the OB domain of the single stranded DNA-binding protein hSSB1 (NABP2/OBFC2B) and chemical shift mapping of the DNA-binding interface, Biomol. NMR Assign. 10 (2) (2016) 297–300. [85] C. Touma, R. Kariawasam, A.X. Gimenez, R.E. Bernardo, N.W. Ashton, M.N. Adams, N. Paquet, T.I. Croll, K.J. O’Byrne, D.J. Richard, L. Cubeddu, R. Gamsjaeger, A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution, Nucleic Acids Res. 44 (16) (2016) 7963–7973. [86] R. Gamsjaeger, R. Kariawasam, Adrian X. Gimenez, C. Touma, E. McIlwain, Ray E. Bernardo, Nicholas E. Shepherd, Sandro F. Ataide, Q. Dong, Derek J. Richard, Malcolm F. White, L. Cubeddu, The structural basis of DNA binding by the single-stranded DNA-binding protein from sulfolobus solfataricus, Biochem. J. 465 (2) (2015) 337–346. [87] H.E. Bryant, N. Schultz, H.D. Thomas, K.M. Parker, D. Flower, E. Lopez, S. Kyle, M. Meuth, N.J. Curtin, T. Helleday, Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase, Nature 434 (7035) (2005) 913–917. [88] H. Farmer, N. McCabe, C.J. Lord, A.N. Tutt, D.A. Johnson, T.B. Richardson, M. Santarosa, K.J. Dillon, I. Hickson, C. Knights, N.M. Martin, S.P. Jackson, G.C. Smith, A. Ashworth, Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy, Nature 434 (7035) (2005) 917–921. [89] K.V. Clark-Knowles, A.M. O’Brien, J.I. Weberpals, BRCA1 as a therapeutic target in sporadic epithelial ovarian cancer, J. Oncol. (2010) (2010), 891059.

Please cite this article in press as: L.V. Croft, et al., Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.03.014