**5.1 Interactive role of RPA and SSB in the unstressed cell cycle**

RPA plays a critical role in replication of DNA and was first identified as a factor for replication of the Simian Virus 40 (SV40). Several reviews provide great detail about the role of RPA in replication; however, most current work examines its function in DNA repair (Fanning, et al., 2006; Iftode, et al., 1999; Wold, 1997). Nevertheless, it is important to understand the role of RPA in normal DNA replication as the switch between its replication role and its repair role is of great interest.

During replication, RPA functions to protect ssDNA and direct the assembly of the replication machinery. In SV40 replication, RPA interacts with T-antigen to facilitate unwinding of the replication origin, through ssDNA stabilization as well as DNA-duplex melting (Georgaki & Hubscher, 1993; Georgaki, et al., 1992; Iftode, et al., 1999; Wold, 1997). Interestingly, nearly all RPAs and SSBs are able to replace human RPA in this role, indicating that stability of the ssDNA intermediate is critical for progression of replication (Wold, 1997). In addition, RPA and SSB serve to direct the formation of the replication complex through direct protein–protein interactions (Binz, et al., 2006; Naue, et al., 2011; Shereda, et al., 2008; Witte, et al., 2003; Wold, 1997; Yuzhakov, Kelman, Hurwitz, et al., 1999).

RPA spatially and temporally directs the addition of key proteins of the replication complex at the site of replication. Temporal control is obtained through competitive binding of the replication components to RPA. Initially, the primase complex is directed to the ssDNA template. Following primer synthesis, Replication Factor C (RFC) binds RPA and loads the replicative clamp PCNA. DNA polymerase δ then binds RPA and is loaded at the DNAprimer junction, allowing DNA replication to begin (Yuzhakov, et al., 1999). These proteinprotein interactions are RPA-specific; SSB cannot substitute, underscoring the important dual role of RPA in both ssDNA binding and protein interactions (Wold, 1997).

Though RPA and SSB cannot always substitute for each other, they play similar roles in the systems in which they reside. Like RPA, SSB serves not only to stabilize ssDNA but also to direct assembly of the replication complex proteins. Through interactions with the Cterminus of SSB, the primase is loaded and retained at the priming site. This interaction may be facilitated through further interactions of SSB with DnaB. This binding is disrupted by the χ subunit of the replicative DNA polymerase III through a multi-step handoff mechanism (Sharma, et al., 2009; Shereda, et al., 2008; Witte, et al., 2003; Yuzhakov, Kelman, & O'Donnell, 1999). Loss of these protein-protein interactions results in cellular demise.

Role of RPA Proteins in Radiation Repair and Recovery 183

replaced with undamaged DNA (Krokan, et al., 2000). RPA physically interacts with and regulates multiple BER glycosylases, including NEIL1 and Ung2, to facilitate correct repair of lesions, in part by inhibiting excisions on ssDNA and promoting incisions on duplex DNA (Mer, et al., 2000; Theriot, et al., 2010). Similarly, SSB interacts with the glycosylase UDG, stimulating or inhibiting its excision activity, depending on the DNA structure at the lesion (Kumar & Varshney, 1997). Nucleotide excision repair (NER) processes remove helixdistorting lesions by excising a patch of DNA, followed by re-synthesis. In this case, RPA physically interacts with the NER mediator XPA to direct incision to the damaged strand, and to help prevent excessive incision events, which could lead to further degradation of the genome (Krasikova, et al., 2010; Overmeer, et al., 2011; Saijo, et al., 2011). In mismatch repair (MMR), RPA and SSB interact with the exonuclease EXO1 to stimulate excision processes, as well as ensure correct excision termination (Genschel & Modrich, 2009; Lu & Keck, 2008).

**5.3 Interactive role of RPA and SSB in the recovery of stalled replication forks** 

Suhasini, et al., 2009; Woodman, et al., 2010; Yuan, et al., 2009; Yusufzai, et al., 2009).

When the replicative helicase is uncoupled from the replisome, large tracts of ssDNA are formed. In eukaryotes this lengthy ssDNA is rapidly coated with RPA, which serves as a signal of replication stress to activate the S phase checkpoint kinase ATR (ATM and Rad3 related), a member of the phosphatidylinositol 3-kinase-like kinase (PIKK) family (Byun, et al., 2005; J. S. Liu, et al., 2006; Zou & Elledge, 2003). In bacteria, the ssDNA is similarly coated with SSB, but a strict cell-cycle control response is absent. ATR is found in a complex with its activator ATRIP (ATR interacting protein) and recruitment of ATR/ATRIP to sites of replication stress is mediated by direct interaction between the N-terminus of RPA1 (DBD F) and ATRIP. This interaction is sufficient for localization of ATR/ATRIP to the stalled replication fork, but activation of ATR requires further protein interactions, also mediated by RPA (X. Xu, et al., 2008; Zou & Elledge, 2003). The Rad17 complex, which comprises Rad17 and RFC subunits 2-5, loads the Rad9-Rad1-Hus1 (9-1-1) DNA damage checkpoint clamp at damage sites. To facilitate loading of the 9-1-1 complex, the Rad17 complex is recruited to damage sites in an RPA-dependent manner (Majka, et al., 2006; Zou, et al., 2003). Rad9 of the 9-1-1 complex binds topoisomerase 2 binding protein 1 (TopBP1), another factor for ATR activation. Simultaneously, Rad9 binds the N-terminus of RPA1 which correctly orients TopBP1 for activation of ATR. Rad9 and ATRIP compete for binding of the N-terminus of RPA1, but each must be bound for efficient activation of ATR. This suggests a model in which

An advancing replication fork must traverse a myriad of DNA lesions through a normal replicative cycle, and the abundance of these lesions increases dramatically under DNAdamaging conditions. Depletion of the nucleotide pool or a single-strand lesion blocks progression of the polymerase complex, while the replicative helicase uncouples from the replisome and continues to unwind template DNA. Such uncoupling results in long segments of ssDNA to which RPA or SSB may bind and actively participate in restart of the stalled fork (Atkinson & McGlynn, 2009; Byun, et al., 2005; McInerney & O'Donnell, 2007; Pages & Fuchs, 2003). Additionally, lesions such as inter-strand crosslinks or covalently linked proteins block advancement of the entire replisome. Such stalls may be repaired and restarted through a fork regression pathway. Several competing fork regression models exist, but all of them require protein elements that closely function and interact with RPA and SSB (Atkinson & McGlynn, 2009; Dronkert & Kanaar, 2001; Machwe, et al., 2011; Shereda, et al., 2007; Shereda, et al., 2008; Sowd, et al., 2009; Sugiyama & Kantake, 2009;

Integral to the role of RPA in DNA replication is its cell-cycle dependent phosphorylation/ dephosphorylation (Figure 2). Specifically, RPA2 is phosphorylated at two consensus sites for Cdc2/Cdk2, resulting in three forms of RPA: unphosphorylated, and two distinct phosphorylated forms (Iftode, et al., 1999; J. S. Liu, et al., 2006; Oakley, et al., 2003; Patrick, et al., 2005; Wold, 1997; Zernik-Kobak, et al., 1997). The majority of RPA is unphosphorylated during G1 phase, and is phosphorylated at either S23 or S29 of RPA2 at the G1/S phase boundary, which is maintained through S phase and into G2 (Anantha, et al., 2007; Oakley, et al., 2003; Stephan, et al., 2009). Near the G2/M phase boundary, a mitotic phosphorylation form becomes abundant (Oakley, et al., 2003; Zernik-Kobak, et al., 1997). This is presumably due to further phosphorylation of RPA2 at the remaining Cdc2/Cdk2 consensus site, which may regulate the ability of RPA to interact with and bind other proteins, thereby modulating its functional role through the cell-cycle (Oakley, et al., 2003; Stephan, et al., 2009). However, the effects of S phase phosphorylation of RPA have not yet been determined, as its ability to support replication and repair are unchanged (Henricksen, et al., 1996; Oakley, et al., 2003; Pan, et al., 1995). In contrast, M phase phosphorylation of RPA abrogates binding to DNA polymerase α, as well as to the checkpoint proteins ATM and DNA-PK (DNA-dependent protein kinase). In addition, its ability to bind duplex DNA is significantly reduced. These modifications may serve to switch RPA away from its replicative role after S phase (Oakley, et al., 2003; Stephan, et al., 2009). It has also been demonstrated that SSB is phosphorylated on tyrosine residues, but the consequences of this phosphorylation remain to be elucidated (Mijakovic, et al., 2006). As such, phosphorylation will be discussed only in relation to RPA for the remainder of this review. Nevertheless, it is reasonable to hypothesize that phosphorylation of SSB plays a similar regulatory role.

Fig. 2. (A) Phosphorylation sites on the N-terminus of RPA2. DNA-PK, Cdc2/Cdk2 and ATR/ATM phosphorylate different sites, which depends on the type of damage and point in the cell-cycle. (B) Proposed mechanism for phosphoregulation of RPA1 N-terminal heterologous protein interactions. Upon phosphorylation of RPA2, the basic N-terminus of RPA1 binds phosphorylated RPA2 and abrogates heterologous protein binding. Protein X represents any of a number of RPA1-interacting proteins.

#### **5.2 Interactive role of RPA and SSB in NER, BER and MMR**

Cellular DNA is under a constant barrage of damaging agents, which may cause crosslinking, base modification or base loss. In general, these types of damage may be repaired by excising the offending lesion and re-synthesizing DNA. RPA and SSB play critical roles in the coordination of these processes. Base excision repair (BER) processes remove damaged bases, forming apurinic/apyrimidinic sites, which are removed and

Integral to the role of RPA in DNA replication is its cell-cycle dependent phosphorylation/ dephosphorylation (Figure 2). Specifically, RPA2 is phosphorylated at two consensus sites for Cdc2/Cdk2, resulting in three forms of RPA: unphosphorylated, and two distinct phosphorylated forms (Iftode, et al., 1999; J. S. Liu, et al., 2006; Oakley, et al., 2003; Patrick, et al., 2005; Wold, 1997; Zernik-Kobak, et al., 1997). The majority of RPA is unphosphorylated during G1 phase, and is phosphorylated at either S23 or S29 of RPA2 at the G1/S phase boundary, which is maintained through S phase and into G2 (Anantha, et al., 2007; Oakley, et al., 2003; Stephan, et al., 2009). Near the G2/M phase boundary, a mitotic phosphorylation form becomes abundant (Oakley, et al., 2003; Zernik-Kobak, et al., 1997). This is presumably due to further phosphorylation of RPA2 at the remaining Cdc2/Cdk2 consensus site, which may regulate the ability of RPA to interact with and bind other proteins, thereby modulating its functional role through the cell-cycle (Oakley, et al., 2003; Stephan, et al., 2009). However, the effects of S phase phosphorylation of RPA have not yet been determined, as its ability to support replication and repair are unchanged (Henricksen, et al., 1996; Oakley, et al., 2003; Pan, et al., 1995). In contrast, M phase phosphorylation of RPA abrogates binding to DNA polymerase α, as well as to the checkpoint proteins ATM and DNA-PK (DNA-dependent protein kinase). In addition, its ability to bind duplex DNA is significantly reduced. These modifications may serve to switch RPA away from its replicative role after S phase (Oakley, et al., 2003; Stephan, et al., 2009). It has also been demonstrated that SSB is phosphorylated on tyrosine residues, but the consequences of this phosphorylation remain to be elucidated (Mijakovic, et al., 2006). As such, phosphorylation will be discussed only in relation to RPA for the remainder of this review. Nevertheless, it is reasonable to hypothesize that phosphorylation of SSB plays a similar regulatory role.

Fig. 2. (A) Phosphorylation sites on the N-terminus of RPA2. DNA-PK, Cdc2/Cdk2 and ATR/ATM phosphorylate different sites, which depends on the type of damage and point in the cell-cycle. (B) Proposed mechanism for phosphoregulation of RPA1 N-terminal heterologous protein interactions. Upon phosphorylation of RPA2, the basic N-terminus of RPA1 binds phosphorylated RPA2 and abrogates heterologous protein binding. Protein X

Cellular DNA is under a constant barrage of damaging agents, which may cause crosslinking, base modification or base loss. In general, these types of damage may be repaired by excising the offending lesion and re-synthesizing DNA. RPA and SSB play critical roles in the coordination of these processes. Base excision repair (BER) processes remove damaged bases, forming apurinic/apyrimidinic sites, which are removed and

represents any of a number of RPA1-interacting proteins.

**5.2 Interactive role of RPA and SSB in NER, BER and MMR** 

replaced with undamaged DNA (Krokan, et al., 2000). RPA physically interacts with and regulates multiple BER glycosylases, including NEIL1 and Ung2, to facilitate correct repair of lesions, in part by inhibiting excisions on ssDNA and promoting incisions on duplex DNA (Mer, et al., 2000; Theriot, et al., 2010). Similarly, SSB interacts with the glycosylase UDG, stimulating or inhibiting its excision activity, depending on the DNA structure at the lesion (Kumar & Varshney, 1997). Nucleotide excision repair (NER) processes remove helixdistorting lesions by excising a patch of DNA, followed by re-synthesis. In this case, RPA physically interacts with the NER mediator XPA to direct incision to the damaged strand, and to help prevent excessive incision events, which could lead to further degradation of the genome (Krasikova, et al., 2010; Overmeer, et al., 2011; Saijo, et al., 2011). In mismatch repair (MMR), RPA and SSB interact with the exonuclease EXO1 to stimulate excision processes, as well as ensure correct excision termination (Genschel & Modrich, 2009; Lu & Keck, 2008).

#### **5.3 Interactive role of RPA and SSB in the recovery of stalled replication forks**

An advancing replication fork must traverse a myriad of DNA lesions through a normal replicative cycle, and the abundance of these lesions increases dramatically under DNAdamaging conditions. Depletion of the nucleotide pool or a single-strand lesion blocks progression of the polymerase complex, while the replicative helicase uncouples from the replisome and continues to unwind template DNA. Such uncoupling results in long segments of ssDNA to which RPA or SSB may bind and actively participate in restart of the stalled fork (Atkinson & McGlynn, 2009; Byun, et al., 2005; McInerney & O'Donnell, 2007; Pages & Fuchs, 2003). Additionally, lesions such as inter-strand crosslinks or covalently linked proteins block advancement of the entire replisome. Such stalls may be repaired and restarted through a fork regression pathway. Several competing fork regression models exist, but all of them require protein elements that closely function and interact with RPA and SSB (Atkinson & McGlynn, 2009; Dronkert & Kanaar, 2001; Machwe, et al., 2011; Shereda, et al., 2007; Shereda, et al., 2008; Sowd, et al., 2009; Sugiyama & Kantake, 2009; Suhasini, et al., 2009; Woodman, et al., 2010; Yuan, et al., 2009; Yusufzai, et al., 2009).

When the replicative helicase is uncoupled from the replisome, large tracts of ssDNA are formed. In eukaryotes this lengthy ssDNA is rapidly coated with RPA, which serves as a signal of replication stress to activate the S phase checkpoint kinase ATR (ATM and Rad3 related), a member of the phosphatidylinositol 3-kinase-like kinase (PIKK) family (Byun, et al., 2005; J. S. Liu, et al., 2006; Zou & Elledge, 2003). In bacteria, the ssDNA is similarly coated with SSB, but a strict cell-cycle control response is absent. ATR is found in a complex with its activator ATRIP (ATR interacting protein) and recruitment of ATR/ATRIP to sites of replication stress is mediated by direct interaction between the N-terminus of RPA1 (DBD F) and ATRIP. This interaction is sufficient for localization of ATR/ATRIP to the stalled replication fork, but activation of ATR requires further protein interactions, also mediated by RPA (X. Xu, et al., 2008; Zou & Elledge, 2003). The Rad17 complex, which comprises Rad17 and RFC subunits 2-5, loads the Rad9-Rad1-Hus1 (9-1-1) DNA damage checkpoint clamp at damage sites. To facilitate loading of the 9-1-1 complex, the Rad17 complex is recruited to damage sites in an RPA-dependent manner (Majka, et al., 2006; Zou, et al., 2003). Rad9 of the 9-1-1 complex binds topoisomerase 2 binding protein 1 (TopBP1), another factor for ATR activation. Simultaneously, Rad9 binds the N-terminus of RPA1 which correctly orients TopBP1 for activation of ATR. Rad9 and ATRIP compete for binding of the N-terminus of RPA1, but each must be bound for efficient activation of ATR. This suggests a model in which

Role of RPA Proteins in Radiation Repair and Recovery 185

RPA focus formation in regression of forks stalled by interstrand crosslinks (Huang, et al.,

Many studies support the hypothesis that RPA directs activity of helicases in fork regression. In eukaryotes, RPA interacts with the RecQ-like helicases WRN and BLM, which have been implicated in the regression of stalled replication forks *in vitro*. RecQ helicases are important for maintenance of chromosome stability; it is therefore not unexpected that they would be involved in fork regression. The interaction of RPA with these proteins may also recruit them to stalled forks; regardless, it stimulates their helicase activity (Brosh, et al., 2000; Brosh, et al., 1999; Doherty, et al., 2005; Machwe, et al., 2011; Machwe, et al., 2006; Machwe, et al., 2007; Ralf, et al., 2006). RPA also recruits the annealing helicase HARP to stalled forks through direct interaction. HARP has been implicated in stabilization and restart of stalled replication forks and may be well suited to assisting fork regression in which complementary DNA strands must be annealed to form stable structures (Ciccia, et al., 2009; Yuan, et al., 2009; Yusufzai, et al., 2009). Thus, the RecQ-like helicases may work with the HARP helicases in the regression of stalled forks. In addition to the human RecQlike helicases, the archaeal RecQ-like helicase Hel308 is also recruited by RPA and is capable of fork regression *in vitro* (Li, et al., 2008; Woodman, et al., 2010). Additionally, the FANCM protein, which is involved in fork regression, interacts with RPA at stalled forks to promote

In bacterial fork regression, similar interactions between SSB and helicases have been observed. SSB binds RecQ through its C-terminus *in vitro* and stimulates RecQ helicase activity (Lecointe, et al., 2007; Shereda, et al., 2007; Shereda, et al., 2008). RecG helicase not only interacts with SSB through the C-terminus but is stabilized by this interaction and supports fork regression (Buss, et al., 2008; Lecointe, et al., 2007; Shereda, et al., 2008). The replication restart helicase PriA also interacts with SSB, which would complete the fork regression process and initiate restart of replication (Cadman & McGlynn, 2004; Kozlov, et al., 2010; Lecointe, et al., 2007; Shereda, et al., 2008). In addition to helicases, SSB interacts with and actively directs the activity of several recombination proteins necessary for the repair of regressed forks before they may be restarted. These processes are essential to other aspects of DNA repair as well, including double-strand break (DSB) repair, discussed below.

The types of DNA damage discussed previously may be repaired directly through NER or in response to replication fork stall. A far more grave form of DNA damage is the DSB, arising from either exogenous or endogenous stresses. A well-known exogenous physical stress that causes DSBs is IR, which is the major reason that it is so detrimental to living systems. A variety of chemical agents, such as bleomycin, phleomycin, and mitomycin C, also act by inducing DSBs. Additionally, a stalled replication fork that is unrepaired may collapse into a special type of DSB with a single double-strand end (Allen, et al., 2011). Regardless, these DSBs are all repaired via similar pathways, which rely on RPA and SSB. Until recently, models of eukaryotic DSB repair indicated that the conserved DNA repair protein complex MRN (Mre11-Rad50-Nbs1) was the first responder and signaler at the site of a DSB. However, recent evidence implicates the novel ssDNA binding protein hSSB1 as the DSB-recognition protein in humans. At the site of damage this protein may bind short ssDNA overhangs and provide protection from incorrect end-processing by nucleases. Additionally, hSSB1 is found complexed with MRN independent of damage, through an

2010). For a review of fork regression models, see Atkinson and McGlynn, 2009.

formation of RPA foci (Banerjee, et al., 2008; Huang, et al., 2010).

**5.4 Interactive role of RPA and SSB in DNA double-strand break repair** 

two adjacent RPAs are required to be bound to ssDNA at a damage site (X. Xu, et al., 2008). This model is further supported by studies describing a ssDNA length-dependent activation of ATR via RPA (J. S. Liu, et al., 2006; Zou & Elledge, 2003).

Activated ATR initiates the intra-S phase checkpoint signal cascade resulting in cell-cycle arrest, stabilization of replication forks, and initiation of damage repair through phosphorylation of effector molecules, including the checkpoint kinase Chk1 (Cimprich & Cortez, 2008; Kastan & Bartek, 2004; Yang, et al., 2003). Activation also leads ATR to phosphorylate S33, a PIKK consensus site, of the RPA2 subunit of ssDNA-bound RPA, which stimulates the Cdc2/Cdk2 kinases to phosphorylate their consensus sites on RPA2 if not already phosphorylated. This, in turn, stimulates phosphorylation of T21 by DNA-PK, which leads to further phosphorylation of RPA2 at residues S4, S8, S11, S12, and S13 by DNA-PK (Anantha, et al., 2007; Vassin, et al., 2009; Zernik-Kobak, et al., 1997). Such hyperphosphorylation of RPA is the major switch from its role in normal uninterrupted replication to an activator and director of DNA damage repair.

Hyperphosphorylation inhibits replicative functions of RPA while stimulating repair and fork stabilization, likely by modifying its protein interaction and duplex DNA binding and destabilizing capabilities. In a rather elegant regulatory mechanism, the phosphorylated RPA2 competitively binds the basic N-terminal region of RPA1, displacing proteins from one of the primary binding surfaces and modulating its interaction capability (Figure 2) (Oakley, et al., 2009). Competitive binding and conformational changes such as this may be responsible for many of the protein interactions that are altered upon phosphorylation of RPA2. Hyperphosphorylation abolishes interaction of RPA with DNA polymerase α, as well as decreases its duplex unwinding ability, which is critical for replication (Oakley, et al., 2003; Patrick, et al., 2005; Wold, 1997). Additionally, in an SV40 *in vitro* system, hyperphosphorylation of RPA leads to a 50% reduction in replication, which can be rescued by unphosphorylated RPA (Patrick, et al., 2005). Hyperphosphorylation, coupled with other aspects of the intra-S phase checkpoint, effectively halts chromosomal replication. However, other RPA-dependent processes, such as damage-dependent DNA synthesis, are unchanged or stimulated. Hyperphosphorylated RPA stimulates *in vivo* synthesis of DNA on RPAcoated ssDNA via a currently unidentified repair polymerase, minimizing ssDNA accumulation during replication stress (Vassin, et al., 2009). Additionally, essential interactions between RPA and NER proteins are unaffected by hyperphosphorylation of RPA (see above) (Oakley, et al., 2003; Patrick, et al., 2005). Many of these repair proteins are capable of interacting with the unphosphorylated C-terminus of RPA2 rather than the Nterminus of RPA1, which may explain the separation of functions between unphosphorylated and hyperphosphorylated RPA (Ali, et al., 2010; Saijo, et al., 2011). Regardless of the mechanism, this demonstrates the versatility of RPA in DNA metabolism. The previous discussion has focused on a restart and repair pathway for replication fork stalls in which the replicative helicase uncouples from the replisome. A replisome may also encounter double-strand lesions, such as interstrand crosslinks, that prevent progression of the entire complex. These lesions, as well as some ssDNA lesions, result in stalled forks that may be repaired and restarted though fork regression. Among the most important proteins in fork regression are repair helicases including those in the RecQ, RecG, HARP, and FANC families. These helicases may perform the complex winding and unwinding of nascent and template strands necessary to form a Holliday junction-like structure referred to as a "chicken foot." Additionally, proteins of the Fanconi anemia pathway may be important for

two adjacent RPAs are required to be bound to ssDNA at a damage site (X. Xu, et al., 2008). This model is further supported by studies describing a ssDNA length-dependent activation of

Activated ATR initiates the intra-S phase checkpoint signal cascade resulting in cell-cycle arrest, stabilization of replication forks, and initiation of damage repair through phosphorylation of effector molecules, including the checkpoint kinase Chk1 (Cimprich & Cortez, 2008; Kastan & Bartek, 2004; Yang, et al., 2003). Activation also leads ATR to phosphorylate S33, a PIKK consensus site, of the RPA2 subunit of ssDNA-bound RPA, which stimulates the Cdc2/Cdk2 kinases to phosphorylate their consensus sites on RPA2 if not already phosphorylated. This, in turn, stimulates phosphorylation of T21 by DNA-PK, which leads to further phosphorylation of RPA2 at residues S4, S8, S11, S12, and S13 by DNA-PK (Anantha, et al., 2007; Vassin, et al., 2009; Zernik-Kobak, et al., 1997). Such hyperphosphorylation of RPA is the major switch from its role in normal uninterrupted

Hyperphosphorylation inhibits replicative functions of RPA while stimulating repair and fork stabilization, likely by modifying its protein interaction and duplex DNA binding and destabilizing capabilities. In a rather elegant regulatory mechanism, the phosphorylated RPA2 competitively binds the basic N-terminal region of RPA1, displacing proteins from one of the primary binding surfaces and modulating its interaction capability (Figure 2) (Oakley, et al., 2009). Competitive binding and conformational changes such as this may be responsible for many of the protein interactions that are altered upon phosphorylation of RPA2. Hyperphosphorylation abolishes interaction of RPA with DNA polymerase α, as well as decreases its duplex unwinding ability, which is critical for replication (Oakley, et al., 2003; Patrick, et al., 2005; Wold, 1997). Additionally, in an SV40 *in vitro* system, hyperphosphorylation of RPA leads to a 50% reduction in replication, which can be rescued by unphosphorylated RPA (Patrick, et al., 2005). Hyperphosphorylation, coupled with other aspects of the intra-S phase checkpoint, effectively halts chromosomal replication. However, other RPA-dependent processes, such as damage-dependent DNA synthesis, are unchanged or stimulated. Hyperphosphorylated RPA stimulates *in vivo* synthesis of DNA on RPAcoated ssDNA via a currently unidentified repair polymerase, minimizing ssDNA accumulation during replication stress (Vassin, et al., 2009). Additionally, essential interactions between RPA and NER proteins are unaffected by hyperphosphorylation of RPA (see above) (Oakley, et al., 2003; Patrick, et al., 2005). Many of these repair proteins are capable of interacting with the unphosphorylated C-terminus of RPA2 rather than the Nterminus of RPA1, which may explain the separation of functions between unphosphorylated and hyperphosphorylated RPA (Ali, et al., 2010; Saijo, et al., 2011). Regardless of the mechanism, this demonstrates the versatility of RPA in DNA metabolism. The previous discussion has focused on a restart and repair pathway for replication fork stalls in which the replicative helicase uncouples from the replisome. A replisome may also encounter double-strand lesions, such as interstrand crosslinks, that prevent progression of the entire complex. These lesions, as well as some ssDNA lesions, result in stalled forks that may be repaired and restarted though fork regression. Among the most important proteins in fork regression are repair helicases including those in the RecQ, RecG, HARP, and FANC families. These helicases may perform the complex winding and unwinding of nascent and template strands necessary to form a Holliday junction-like structure referred to as a "chicken foot." Additionally, proteins of the Fanconi anemia pathway may be important for

ATR via RPA (J. S. Liu, et al., 2006; Zou & Elledge, 2003).

replication to an activator and director of DNA damage repair.

RPA focus formation in regression of forks stalled by interstrand crosslinks (Huang, et al., 2010). For a review of fork regression models, see Atkinson and McGlynn, 2009.

Many studies support the hypothesis that RPA directs activity of helicases in fork regression. In eukaryotes, RPA interacts with the RecQ-like helicases WRN and BLM, which have been implicated in the regression of stalled replication forks *in vitro*. RecQ helicases are important for maintenance of chromosome stability; it is therefore not unexpected that they would be involved in fork regression. The interaction of RPA with these proteins may also recruit them to stalled forks; regardless, it stimulates their helicase activity (Brosh, et al., 2000; Brosh, et al., 1999; Doherty, et al., 2005; Machwe, et al., 2011; Machwe, et al., 2006; Machwe, et al., 2007; Ralf, et al., 2006). RPA also recruits the annealing helicase HARP to stalled forks through direct interaction. HARP has been implicated in stabilization and restart of stalled replication forks and may be well suited to assisting fork regression in which complementary DNA strands must be annealed to form stable structures (Ciccia, et al., 2009; Yuan, et al., 2009; Yusufzai, et al., 2009). Thus, the RecQ-like helicases may work with the HARP helicases in the regression of stalled forks. In addition to the human RecQlike helicases, the archaeal RecQ-like helicase Hel308 is also recruited by RPA and is capable of fork regression *in vitro* (Li, et al., 2008; Woodman, et al., 2010). Additionally, the FANCM protein, which is involved in fork regression, interacts with RPA at stalled forks to promote formation of RPA foci (Banerjee, et al., 2008; Huang, et al., 2010).

In bacterial fork regression, similar interactions between SSB and helicases have been observed. SSB binds RecQ through its C-terminus *in vitro* and stimulates RecQ helicase activity (Lecointe, et al., 2007; Shereda, et al., 2007; Shereda, et al., 2008). RecG helicase not only interacts with SSB through the C-terminus but is stabilized by this interaction and supports fork regression (Buss, et al., 2008; Lecointe, et al., 2007; Shereda, et al., 2008). The replication restart helicase PriA also interacts with SSB, which would complete the fork regression process and initiate restart of replication (Cadman & McGlynn, 2004; Kozlov, et al., 2010; Lecointe, et al., 2007; Shereda, et al., 2008). In addition to helicases, SSB interacts with and actively directs the activity of several recombination proteins necessary for the repair of regressed forks before they may be restarted. These processes are essential to other aspects of DNA repair as well, including double-strand break (DSB) repair, discussed below.

#### **5.4 Interactive role of RPA and SSB in DNA double-strand break repair**

The types of DNA damage discussed previously may be repaired directly through NER or in response to replication fork stall. A far more grave form of DNA damage is the DSB, arising from either exogenous or endogenous stresses. A well-known exogenous physical stress that causes DSBs is IR, which is the major reason that it is so detrimental to living systems. A variety of chemical agents, such as bleomycin, phleomycin, and mitomycin C, also act by inducing DSBs. Additionally, a stalled replication fork that is unrepaired may collapse into a special type of DSB with a single double-strand end (Allen, et al., 2011). Regardless, these DSBs are all repaired via similar pathways, which rely on RPA and SSB.

Until recently, models of eukaryotic DSB repair indicated that the conserved DNA repair protein complex MRN (Mre11-Rad50-Nbs1) was the first responder and signaler at the site of a DSB. However, recent evidence implicates the novel ssDNA binding protein hSSB1 as the DSB-recognition protein in humans. At the site of damage this protein may bind short ssDNA overhangs and provide protection from incorrect end-processing by nucleases. Additionally, hSSB1 is found complexed with MRN independent of damage, through an

Role of RPA Proteins in Radiation Repair and Recovery 187

SSB protein does not appear to respond to DNA damage, suggesting that any regulation on this promoter is compensated by opposite regulation on the others (Meyer & Laine, 1990). One of the two homologs in *B. subtilis* functions in natural competence, whereas the gene for the replication SSB is not damage-inducible (Lindner, et al., 2004). The promoters for both the canonical SSB and the newly identified DdrB in *D. radiodurans* contain a recognizable "radiation-desiccation response motif"; both genes are up-regulated in response to IR, but *ssb* is not induced by desiccation or UV (Ujaoney, et al., 2010). The SSB protein is present in *D. radiodurans* at ten-fold higher levels than in *E. coli*, and, unlike in *E. coli*, levels increase 4 fold after irradiation (Bernstein, et al., 2004; Y. Liu, et al., 2003). In contrast, the transcript for the *ddrB* gene is normally almost absent, but is induced 40-fold within minutes after exposure to IR (Tanaka, et al., 2004). The IR-sensitivity, and viability, of *ddrB* knockouts

In yeast and humans, the presence of a single RPA makes transcriptional regulation in response to damage less likely. In yeast, expression of RPA is cell-cycle dependent, with maximal expression prior to S phase (Brill & Stillman, 1991). Human RPA levels remain constant throughout the cell cycle (Wold, 1997). However, the alternative RPA2 subunit, RPA4, is more highly expressed in quiescent cells, and, since it competes with RPA2 for binding to RPA1 and RPA3, must be down-regulated in dividing cells (Haring, et al., 2010). This long-term regulation, however, is presumably not readily reversible, and is not a damage response. Interestingly, RPA1 and RPA2 levels are higher in colon cancer cells but it is not clear how this increase is achieved (Givalos, et al., 2007). Plants have multiple homologs, which are differentially expressed in tissues. There is evidence for regulation of certain homologs in response to DNA damage, as well as in response to certain hormones

The single crenarchaeal RPA homolog would not be expected to be transcriptionally regulated to any significant extent, given its essential role in replication. In the euryarchaea, however, where multiple homologs are common, regulation has been reported. The transcriptome of *H. salinarum* during recovery from exposure to IR or UV has been analyzed and only one of the five RPA homologs showed any any increase in expression (Baliga, et al., 2004; Kottemann, et al., 2005; McCready, et al., 2005). This homolog, encoded by the *rfa3* operon, is one of two eukaryotic-like RPAs present in *H. salinarum*. Constitutive upregulation of this operon was found as the only transcriptomic difference among multiple radiation-resistant mutants of *H. salinarum* (DeVeaux, et al., 2007). This homolog is significantly up-regulated following IR-treatment of wild type *H. salinarum*, but not in the radiation-resistant mutants, presumably because the protein is being constitutively expressed at the induced level. In addition, both wild-type and mutant strains show induced expression of the second RPA-like homolog, encoded by the *rfa2* operon. Neither of the crenarchaeal nor the euryarchaeal-like SSB/RPA homologs shows significant transcriptional differences after irradiation (Gygli and DeVeaux, unpublished observations). These results suggest that the eukaryotic RPA-like homologs present in this organism may

The highly conserved and essential nature of the RPA/SSB family underscores the fundamental role that these proteins play in basic DNA metabolic processes. The interactions that these proteins are involved in illustrate an intricate network and temporal

underscores its specific role in DNA repair, and not replication.

(Marwedel, et al., 2003; Sakaguchi, et al., 2009).

be more directly involved in repair of radiation damage.

**7. Conclusion** 

interaction between the C-terminal tail of hSSB1 and the N-terminal region of Nbs1. This direct interaction, along with the ssDNA-bound hSSB1, stimulates the recruitment of the MRN complex to DSB sites. RPA cannot replace hSSB1 in this function (Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011). hSSB1 may also have duplex melting capabilities similar to that seen in other SSBs, which would further help to provide a substrate for MRN binding (Cubeddu & White, 2005; Eggington, et al., 2006; Richard, Cubeddu, et al., 2011).

Once recruited to a DSB, the MRN complex tethers the broken DNA ends; its endonuclease activity is stimulated through hSSB1 association. MRN then activates the ATM kinase, which in turn initiates a phosphorylation signal cascade similar to ATR. Among the many phosphorylation targets of ATM are the cell-cycle checkpoint kinase Chk2, histone variant H2AX, and hSSB1 (Czornak, et al., 2008; Richard, et al., 2008; Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011; Williams, et al., 2010). Phosphorylation of Chk2 results in cellcycle arrest, while phosphorylated H2AX serves as a local binding point for repair proteins (Kastan & Bartek, 2004; J. E. Kim, et al., 2006). hSSB1 phosphorylation has unknown consequences, but it is likely to alter its repair role in a manner similar to the phosphoregulation of RPA (Richard, et al., 2008; Richard, Savage, et al., 2011). The nuclease activity of MRN results in limited end-resection at the DSB providing a substrate to which RPA may then bind (Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011). ssDNA-bound RPA activates the ATR response and may also be phosphorylated by either activated ATM or ATR, switching it to a repair-process director (Jazayeri, et al., 2006; Oakley, et al., 2001).

In both eukaryotic and bacterial systems, DSB repair and fork regression may proceed via homologous recombination when a homologous template is present. Not surprisingly, RPA and SSB play central roles in this process as well. The RPA or SSB coating ssDNA at the site of damage is replaced with recombinase Rad51 or RecA, respectively, through direct interaction with mediators of homologous recombination. Specifically, the replacement of RPA by Rad51 is facilitated by interactions between the N-terminus of RPA1 and the homologous recombination mediator Rad52 (Plate, et al., 2008; Sugiyama & Kantake, 2009). Akin to requirements for ATR activation, multiple RPAs are necessary to promote this exchange. Similarly, bacterial SSB interacts through its C-terminal tail with the recombination mediator RecO to facilitate loading of RecA onto ssDNA by RecFOR (Costes, et al., 2010; Inoue, et al., 2011). Another interesting interaction of SSB is its apparent competition with the RecA inhibitor RecX for the common ssDNA ligand. By outcompeting RecX for access to ssDNA, SSB prevents RecX from inhibiting RecA loading onto the ssDNA (Baitin, et al., 2008). Interactions between archaeal SSB and a RadA-mediator Rad55 have also been demonstrated *in vitro*. This interaction promotes loading of RadA onto ssDNA suggesting a similar function to that seen in eukaryotes and bacteria (Sheng, et al., 2008).

#### **6. Transcriptional regulation**

The post-translational modifications of RPA/SSB provide virtually instantaneous response to conditions within the cell. Less well-documented, but potentially as important to survival, is transcriptional regulation of the genes in response to more global DNA damage. There is evidence of such regulation in all three domains of life. In general, where damage-induced transcriptional regulation has been reported, the organism contains multiple homologs, and the damage-regulated RPA is generally not required for replication.

In bacteria, the well-studied *E. coli ssb* gene contains multiple promoters, one of which has been shown to be damage-inducible (Brandsma, et al., 1985). Despite this, the level of *E. coli*

interaction between the C-terminal tail of hSSB1 and the N-terminal region of Nbs1. This direct interaction, along with the ssDNA-bound hSSB1, stimulates the recruitment of the MRN complex to DSB sites. RPA cannot replace hSSB1 in this function (Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011). hSSB1 may also have duplex melting capabilities similar to that seen in other SSBs, which would further help to provide a substrate for MRN binding (Cubeddu & White, 2005; Eggington, et al., 2006; Richard, Cubeddu, et al., 2011). Once recruited to a DSB, the MRN complex tethers the broken DNA ends; its endonuclease activity is stimulated through hSSB1 association. MRN then activates the ATM kinase, which in turn initiates a phosphorylation signal cascade similar to ATR. Among the many phosphorylation targets of ATM are the cell-cycle checkpoint kinase Chk2, histone variant H2AX, and hSSB1 (Czornak, et al., 2008; Richard, et al., 2008; Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011; Williams, et al., 2010). Phosphorylation of Chk2 results in cellcycle arrest, while phosphorylated H2AX serves as a local binding point for repair proteins (Kastan & Bartek, 2004; J. E. Kim, et al., 2006). hSSB1 phosphorylation has unknown consequences, but it is likely to alter its repair role in a manner similar to the phosphoregulation of RPA (Richard, et al., 2008; Richard, Savage, et al., 2011). The nuclease activity of MRN results in limited end-resection at the DSB providing a substrate to which RPA may then bind (Richard, Cubeddu, et al., 2011; Richard, Savage, et al., 2011). ssDNA-bound RPA activates the ATR response and may also be phosphorylated by either activated ATM or ATR, switching it to a repair-process director (Jazayeri, et al., 2006; Oakley, et al., 2001). In both eukaryotic and bacterial systems, DSB repair and fork regression may proceed via homologous recombination when a homologous template is present. Not surprisingly, RPA and SSB play central roles in this process as well. The RPA or SSB coating ssDNA at the site of damage is replaced with recombinase Rad51 or RecA, respectively, through direct interaction with mediators of homologous recombination. Specifically, the replacement of RPA by Rad51 is facilitated by interactions between the N-terminus of RPA1 and the homologous recombination mediator Rad52 (Plate, et al., 2008; Sugiyama & Kantake, 2009). Akin to requirements for ATR activation, multiple RPAs are necessary to promote this exchange. Similarly, bacterial SSB interacts through its C-terminal tail with the recombination mediator RecO to facilitate loading of RecA onto ssDNA by RecFOR (Costes, et al., 2010; Inoue, et al., 2011). Another interesting interaction of SSB is its apparent competition with the RecA inhibitor RecX for the common ssDNA ligand. By outcompeting RecX for access to ssDNA, SSB prevents RecX from inhibiting RecA loading onto the ssDNA (Baitin, et al., 2008). Interactions between archaeal SSB and a RadA-mediator Rad55 have also been demonstrated *in vitro*. This interaction promotes loading of RadA onto ssDNA suggesting a similar function to that seen in eukaryotes and bacteria (Sheng, et al., 2008).

The post-translational modifications of RPA/SSB provide virtually instantaneous response to conditions within the cell. Less well-documented, but potentially as important to survival, is transcriptional regulation of the genes in response to more global DNA damage. There is evidence of such regulation in all three domains of life. In general, where damage-induced transcriptional regulation has been reported, the organism contains multiple homologs, and

In bacteria, the well-studied *E. coli ssb* gene contains multiple promoters, one of which has been shown to be damage-inducible (Brandsma, et al., 1985). Despite this, the level of *E. coli*

the damage-regulated RPA is generally not required for replication.

**6. Transcriptional regulation** 

SSB protein does not appear to respond to DNA damage, suggesting that any regulation on this promoter is compensated by opposite regulation on the others (Meyer & Laine, 1990). One of the two homologs in *B. subtilis* functions in natural competence, whereas the gene for the replication SSB is not damage-inducible (Lindner, et al., 2004). The promoters for both the canonical SSB and the newly identified DdrB in *D. radiodurans* contain a recognizable "radiation-desiccation response motif"; both genes are up-regulated in response to IR, but *ssb* is not induced by desiccation or UV (Ujaoney, et al., 2010). The SSB protein is present in *D. radiodurans* at ten-fold higher levels than in *E. coli*, and, unlike in *E. coli*, levels increase 4 fold after irradiation (Bernstein, et al., 2004; Y. Liu, et al., 2003). In contrast, the transcript for the *ddrB* gene is normally almost absent, but is induced 40-fold within minutes after exposure to IR (Tanaka, et al., 2004). The IR-sensitivity, and viability, of *ddrB* knockouts underscores its specific role in DNA repair, and not replication.

In yeast and humans, the presence of a single RPA makes transcriptional regulation in response to damage less likely. In yeast, expression of RPA is cell-cycle dependent, with maximal expression prior to S phase (Brill & Stillman, 1991). Human RPA levels remain constant throughout the cell cycle (Wold, 1997). However, the alternative RPA2 subunit, RPA4, is more highly expressed in quiescent cells, and, since it competes with RPA2 for binding to RPA1 and RPA3, must be down-regulated in dividing cells (Haring, et al., 2010). This long-term regulation, however, is presumably not readily reversible, and is not a damage response. Interestingly, RPA1 and RPA2 levels are higher in colon cancer cells but it is not clear how this increase is achieved (Givalos, et al., 2007). Plants have multiple homologs, which are differentially expressed in tissues. There is evidence for regulation of certain homologs in response to DNA damage, as well as in response to certain hormones (Marwedel, et al., 2003; Sakaguchi, et al., 2009).

The single crenarchaeal RPA homolog would not be expected to be transcriptionally regulated to any significant extent, given its essential role in replication. In the euryarchaea, however, where multiple homologs are common, regulation has been reported. The transcriptome of *H. salinarum* during recovery from exposure to IR or UV has been analyzed and only one of the five RPA homologs showed any any increase in expression (Baliga, et al., 2004; Kottemann, et al., 2005; McCready, et al., 2005). This homolog, encoded by the *rfa3* operon, is one of two eukaryotic-like RPAs present in *H. salinarum*. Constitutive upregulation of this operon was found as the only transcriptomic difference among multiple radiation-resistant mutants of *H. salinarum* (DeVeaux, et al., 2007). This homolog is significantly up-regulated following IR-treatment of wild type *H. salinarum*, but not in the radiation-resistant mutants, presumably because the protein is being constitutively expressed at the induced level. In addition, both wild-type and mutant strains show induced expression of the second RPA-like homolog, encoded by the *rfa2* operon. Neither of the crenarchaeal nor the euryarchaeal-like SSB/RPA homologs shows significant transcriptional differences after irradiation (Gygli and DeVeaux, unpublished observations). These results suggest that the eukaryotic RPA-like homologs present in this organism may be more directly involved in repair of radiation damage.

#### **7. Conclusion**

The highly conserved and essential nature of the RPA/SSB family underscores the fundamental role that these proteins play in basic DNA metabolic processes. The interactions that these proteins are involved in illustrate an intricate network and temporal

Role of RPA Proteins in Radiation Repair and Recovery 189

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