**4. Characterized mutations of RPA and SSB**

*E. coli* SSB mutants were first isolated in a screen for DNA replication mutants (Meyer & Laine, 1990). Although the two best-characterized of these temperature-sensitive (ts) mutations, *ssb-1* and *ssb-113*, fail to grow at 42o C due to inability of the labile SSB to participate in DNA replication, *ssb-1* mutants have essentially normal phenotypes at the permissive temperature. The mutation (H55Y) resides in the OB-fold and affects the ability of the protein to form homotetramers (Shereda, et al., 2008). In contrast, even at the permissive growth temperature, *ssb-113* mutants are severely compromised in their ability to survive numerous DNA damaging agents, and display recombination deficiencies (Chase, et al., 1984). The extremely pleiotropic phenotype conferred by the amino acid change (P176S of 177) in *ssb-113* suggests that the protein interaction capabilities of the C-terminal region are necessary for more than just replication. A protein lacking only 10 amino acids from the Cterminus is non-functional (Curth, et al., 1996). Indeed, mutation of the terminal phenylalanine is lethal, and disrupts protein-protein interactions (Genschel, et al., 2000). These phenotypes provided support not only for the commonality of protein function in recombination, repair, and replication, but also early evidence that these roles were separable within an individual protein. In contrast, a *B. subtilis* mutant in which SSB lacks the Cterminal 35 amino acids, involved in interaction with the helicase PriA, is viable, demonstrating that this conserved region is not essential in all bacteria (Lecointe, et al., 2007). In the budding yeast *S. cerevisiae*, all three subunits of RPA are essential (Brill & Stillman, 1991; Maniar, et al., 1997). Like *E. coli ssb-113* mutants, ts-mutants of RPA1 are profoundly UV- and ionizing radiation (IR)-sensitive at the permissive temperature (Parker, et al., 1997). Several ts-mutants display a mutator phenotype, which is likely related to defects in replication rather than repair (Chen, et al., 1998). Small insertions in either the RPA1 DNAbinding domain or the N-terminal protein-interaction domain result in a marked decrease in survival after UV exposure; however, only the latter is defective in the cell-cycle response to such damage. In addition, only the DNA-binding mutant is defective in repair of UVinduced lesions (Longhese, et al., 1996; Teng, et al., 1998). The N-terminal domain is required for interactions with the clamp loader. This binding is abolished in the *rfa1-t11* mutation, which contains a single change (K45E) (H. S. Kim & Brill, 2001; Majka, et al., 2006; Umezu, et al., 1998). This same allele is deficient in many pathways requiring interactions

Role of RPA Proteins in Radiation Repair and Recovery 181

damaging agents, indicating a role in repair but not replication. Likewise, in *Arabidopsis thaliana*, deletion of one RPA1 homolog results in meiotic defects; although DNA breaks appear to be repaired normally, there is a deficiency in meiotic crossover (Osman, et al., 2009). During vegetative growth, plants containing this mutation are sensitive to DNAdamaging agents (Takashi, et al., 2009). The presence of multiple homologs, some of which are not essential and appear not to be involved with replication, suggests that RPAs in plants have duplicated and diverged, resulting in specialization and separation of function.

While RPAs and SSBs are indispensible for normal replication, they are also central to nearly all DNA repair pathways. The overarching theme for RPA/SSB in these repair pathways is their function as directors of key enzymatic proteins without themselves being enzymatic. The following sections will pertain to the canonical RPA and SSB unless otherwise noted.

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

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,

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

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.

dual role of RPA in both ssDNA binding and protein interactions (Wold, 1997).

**5. Interactive roles of RPA and SSB in DNA metabolism** 

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

role and its repair role is of great interest.

1997; Yuzhakov, Kelman, Hurwitz, et al., 1999).

with DNA processing proteins, including repair of DNA breaks incurred during meiotic recombination, and interaction with Rad51 and its mediator Rad52 (Soustelle, et al., 2002; Sugiyama & Kantake, 2009). Deletion of either the N-terminal 20 amino acids or any deletion at the C-terminus is lethal (Philipova, et al., 1996). In the fission yeast *S. pombe*, as in *S. cerevisiae*, mutations in the large subunit gene confer sensitivity to DNA damaging agents such as UV; in addition, these mutations confer a deficiency in telomere maintenance that is seen in *S. cerevisiae* only in combination with a Ku mutation (Ono, et al., 2003).

In *S. cerevisiae*, mutations in RPA2 also confer sensitivity to various DNA-damaging agents and display defects in replication fidelity (Maniar, et al., 1997; Santocanale, et al., 1995). Tsmutants arrest in S phase at the nonpermissive temperature. Although the protein is essential, the DNA-binding domain is not; only the central protein-interaction domain is required (Dickson, et al., 2009; Philipova, et al., 1996). Temperature-sensitive mutations in RPA3 confer a replication defect in *S. cerevisiae.* Whereas replication ceases immediately in ts-mutants of RPA2, RPA3 mutations allow one round of replication before cessation (Maniar, et al., 1997). As in RPA1, deletions of the RPA3 N-terminus are viable, but Cterminal deletions are not tolerated (Philipova, et al., 1996). Interestingly, RPA3 is not essential in *S. pombe*, and is dispensible in meiosis (Cavero, et al., 2010). However, cells containing a gene deletion show marked sensitivity to DNA damaging agents, particularly those interfering with replication, but not to IR. This suggests that RPA3 in *S. pombe* is involved in repair of replication damage, but not homologous recombination. It is possible that an additional RPA3 homolog will be discovered in *S. pombe* that carries out the essential role in replication performed by a single RPA3 in other organisms.

In human cells, depletion of RPA results not only in increased spontaneous DNA damage and decreased cell viability, but also in asynchrony, arrest at the G1/S boundary, a slowing of progression through S phase, and arrest at the G2/M checkpoint (Dodson, et al., 2004; Haring, et al., 2008). The G2/M arrest was found to be the result of constitutive activation of ATM (ataxia telangiectasia mutated) kinase resulting from lack of RPA. Cells harboring mutations in DBD A, B or C are able to replicate DNA, and traverse S phase, but arrest at the G2/M checkpoint. In contrast to yeast, the N-terminal 168 amino acids of RPA1 are not essential for replication or cell-cycle progression. RPA1 mutants defective in ssDNA binding are still able to support replication; however, some mutants with very modest defects in DNA binding are severely compromised in cell-cycle progression (Haring, et al., 2008). One mutation, L221P, in which a highly conserved leucine in DBD A is changed to proline, has been characterized in yeast (Chen & Kolodner, 1999; Chen, et al., 1998), in mice (Wang, et al., 2005), and in humans (Hass, et al., 2010), and has been shown to promote chromosomal instability. This mutation is lethal in mice; heterozygosity leads to shortened life spans and increased cancer incidence. The conservation of this residue and the drastic phenotype associated with its replacement indicates a critical role of this DNA binding domain in the fundamental role of RPA in replication. A mutation changing another conserved residue in the same domain, D227Y, causes telomere shortening in human cancer cells (Kobayashi, et al., 2010), as does the analogous change in yeast (Ono, et al., 2003*).* Much less is known about mutations of the two smaller subunits in mammalian systems. However, as in yeast, human RPA2 is required for replication, and the only essential region is the central proteininteracting domain (Fanning, et al., 2006; Haring, et al., 2010).

Higher plants contain multiple homologs of at least the large and medium subunits. In rice, one homolog of RPA1 is not essential during vegetative growth, but mutants are sterile, indicating a meiotic defect (Chang, et al., 2009). Also, mutants are sensitive to DNA-

with DNA processing proteins, including repair of DNA breaks incurred during meiotic recombination, and interaction with Rad51 and its mediator Rad52 (Soustelle, et al., 2002; Sugiyama & Kantake, 2009). Deletion of either the N-terminal 20 amino acids or any deletion at the C-terminus is lethal (Philipova, et al., 1996). In the fission yeast *S. pombe*, as in *S. cerevisiae*, mutations in the large subunit gene confer sensitivity to DNA damaging agents such as UV; in addition, these mutations confer a deficiency in telomere maintenance that is

In *S. cerevisiae*, mutations in RPA2 also confer sensitivity to various DNA-damaging agents and display defects in replication fidelity (Maniar, et al., 1997; Santocanale, et al., 1995). Tsmutants arrest in S phase at the nonpermissive temperature. Although the protein is essential, the DNA-binding domain is not; only the central protein-interaction domain is required (Dickson, et al., 2009; Philipova, et al., 1996). Temperature-sensitive mutations in RPA3 confer a replication defect in *S. cerevisiae.* Whereas replication ceases immediately in ts-mutants of RPA2, RPA3 mutations allow one round of replication before cessation (Maniar, et al., 1997). As in RPA1, deletions of the RPA3 N-terminus are viable, but Cterminal deletions are not tolerated (Philipova, et al., 1996). Interestingly, RPA3 is not essential in *S. pombe*, and is dispensible in meiosis (Cavero, et al., 2010). However, cells containing a gene deletion show marked sensitivity to DNA damaging agents, particularly those interfering with replication, but not to IR. This suggests that RPA3 in *S. pombe* is involved in repair of replication damage, but not homologous recombination. It is possible that an additional RPA3 homolog will be discovered in *S. pombe* that carries out the essential

In human cells, depletion of RPA results not only in increased spontaneous DNA damage and decreased cell viability, but also in asynchrony, arrest at the G1/S boundary, a slowing of progression through S phase, and arrest at the G2/M checkpoint (Dodson, et al., 2004; Haring, et al., 2008). The G2/M arrest was found to be the result of constitutive activation of ATM (ataxia telangiectasia mutated) kinase resulting from lack of RPA. Cells harboring mutations in DBD A, B or C are able to replicate DNA, and traverse S phase, but arrest at the G2/M checkpoint. In contrast to yeast, the N-terminal 168 amino acids of RPA1 are not essential for replication or cell-cycle progression. RPA1 mutants defective in ssDNA binding are still able to support replication; however, some mutants with very modest defects in DNA binding are severely compromised in cell-cycle progression (Haring, et al., 2008). One mutation, L221P, in which a highly conserved leucine in DBD A is changed to proline, has been characterized in yeast (Chen & Kolodner, 1999; Chen, et al., 1998), in mice (Wang, et al., 2005), and in humans (Hass, et al., 2010), and has been shown to promote chromosomal instability. This mutation is lethal in mice; heterozygosity leads to shortened life spans and increased cancer incidence. The conservation of this residue and the drastic phenotype associated with its replacement indicates a critical role of this DNA binding domain in the fundamental role of RPA in replication. A mutation changing another conserved residue in the same domain, D227Y, causes telomere shortening in human cancer cells (Kobayashi, et al., 2010), as does the analogous change in yeast (Ono, et al., 2003*).* Much less is known about mutations of the two smaller subunits in mammalian systems. However, as in yeast, human RPA2 is required for replication, and the only essential region is the central protein-

Higher plants contain multiple homologs of at least the large and medium subunits. In rice, one homolog of RPA1 is not essential during vegetative growth, but mutants are sterile, indicating a meiotic defect (Chang, et al., 2009). Also, mutants are sensitive to DNA-

seen in *S. cerevisiae* only in combination with a Ku mutation (Ono, et al., 2003).

role in replication performed by a single RPA3 in other organisms.

interacting domain (Fanning, et al., 2006; Haring, et al., 2010).

damaging agents, indicating a role in repair but not replication. Likewise, in *Arabidopsis thaliana*, deletion of one RPA1 homolog results in meiotic defects; although DNA breaks appear to be repaired normally, there is a deficiency in meiotic crossover (Osman, et al., 2009). During vegetative growth, plants containing this mutation are sensitive to DNAdamaging agents (Takashi, et al., 2009). The presence of multiple homologs, some of which are not essential and appear not to be involved with replication, suggests that RPAs in plants have duplicated and diverged, resulting in specialization and separation of function.
