**2. Structure**

There are two primary classes of ssDNA binding proteins, which share secondary and tertiary structural features, but are distinct in their quaternary structures. The eukaryotic Replication Protein A class (RPAs) consists of heterotrimeric proteins while the bacterial class (SSBs) consists of a range of homo-multimers. Members of the archaeal domain may possess either the RPA or SSB types, or combinations unique to this domain, illustrated in Figure 1 (Kerr, et al., 2003; Lin, et al., 2008; Richard, et al., 2009; Shereda, et al., 2008; Wold, 1997).

SSBs/RPAs are primarily identified by the presence of a structurally conserved oligonucleotide/oligosaccharide binding motif (OB-fold) (Kerr, et al., 2003; Richard, et al., 2009; Shereda, et al., 2008; Theobald, et al., 2003; Wold, 1997). The canonical OB-fold consists of five structurally conserved β-strands, forming two β-sheets, and their inter-spaced variable loops, which form a tertiary flattened β-barrel. The non-specific binding of ssDNA occurs on the surface of the β-barrel in a cleft between the variable loops. The binding of nucleotides is mediated through stacking interactions with aromatic residues and packing interactions with hydrophobic residues. Binding to the phosphodiester backbone also occurs through electrostatic interactions. The OB-folds have a binding polarity that specifies the orientation on the bound ssDNA (Shereda, et al., 2008; Theobald, et al., 2003).

The majority of bacterial and mitochondrial SSBs function as homotetramers, with a single OB-fold per monomer and thus four OB-folds per complex. For members of the *Deinococcus/Thermus* branch, the SSB functions as a homodimer and maintains the theme of four OB-folds per complex by having two non-identical OB-folds per monomer (Bernstein, et al., 2004; Eggington, et al., 2004; Filipkowski, et al., 2006; Filipkowski & Kur, 2007;

Role of RPA Proteins in Radiation Repair and Recovery 177

Fanning, et al., 2006; Oakley, et al., 2009; Wold, 1997). The N-terminus of RPA2 contains a regulatory phosphorylation domain that affects interactions of RPA with other proteins, as well as its DNA binding kinetics (Machwe, et al., 2011; Nuss, et al., 2005; Oakley, et al., 2009; Patrick, et al., 2005; Vassin, et al., 2009). The C-terminal region of RPA2 is also involved in heterologous protein interactions and regulation. RPA3 consists almost entirely of DBD E and is thought to be involved primarily in heterotrimer formation, but potential roles in heterologous protein interactions have been identified (Cavero, et al., 2010; Wold, 1997).

The highly conserved nature of the OB-fold has allowed identification of potential RPA/SSB homologs in the ever-expanding genome sequence database. To our knowledge, there have been no reports of genomes lacking an RPA/SSB homolog, with the possible exception of

There are numerous excellent and extensive reviews covering the history, genetics and biochemical characterization of *E. coli* SSB and its encoding gene (Lohman & Ferrari, 1994; Meyer & Laine, 1990; Shereda, et al., 2008). The single gene, *ssb*, is located adjacent to, but divergently transcribed from, the *uvrA* gene (Brandsma, et al., 1985). In *Bacillus subtilis*, there are two SSB genes, *ssb* and *ywpH* (Lindner, et al., 2004). Although the amino acid sequences of the two proteins are similar, YwpH lacks the C-terminal region found in both the *E. coli* and *B. subtilis* SSBs. *B. subtilis ssb* is essential, but unlike in *E. coli*, is found between ribosomal protein genes. Of 87 bacterial genomes analyzed, all contained at least one SSB gene (Lindner, et al., 2004). Based on gene organization, four groupings were proposed: Group I organisms display *B. subtilis-*type gene organization and more than one SSB. Group II organisms contain only one gene for SSB. *E. coli* represents the type organism for Group III bacteria, where the single *ssb* gene is divergently transcribed from the adjacent *uvrA* gene. Group IV consists of organisms whose SSB organization does not resemble either *B. subtilis* or *E. coli*. *Thermotoga maritima* and *T. neopolitana* likely fall into Group IV, whereas *Thermoanaerobacter tengcongensis* is in Group I (Olszewski, et al., 2010;

Recently, a novel homopentameric SSB, DdrB, was identified in *Deinococcus radiodurans*, which may challenge previous notions regarding canonical SSB structure (Norais, et al., 2009; Sugiman-Marangos & Junop, 2010). Restricted to the Deinococcal lineage, it may represent an adaptation related to the unusually high DNA repair capacity of this organism. However, it should be kept in mind that other SSB proteins that deviate from the traditional

Three separate genes encode the canonical eukaryotic RPA, which is the subject of several recent review articles (Binz, et al., 2004; Broderick, et al., 2010; Richard, et al., 2009; Wold, 1997). However, in higher plants each subunit of RPA may be represented by multiple genes

Both *Saccharomyces cerevisiae* and *Schizosaccharomyces pombe* contain a single gene for each of the three RPA subunits. All three are essential in *S. cerevisiae*, but RPA3 appears to be nonessential in *S. pombe* (Brill & Stillman, 1991; Cavero, et al., 2010; Dickson, et al., 2009; Maniar, et al., 1997). Given that RPA3 is the most variable, and the essential role that the intact protein plays in replication, an additional RPA3 gene may yet be identified that carries out this role in *S. pombe*. In addition to heterotrimeric RPA, a protein complex, Stn1/Ten1, resembling an RPA2/RPA3 dimer has been found to be necessary for telomere maintenance

consensus may yet be identified (Norais, et al., 2009; G. Xu, et al., 2010).

whose proteins function in distinct processes (Sakaguchi, et al., 2009).

the crenarchaea *Pyrobaculum aerophilum* and *Aeropyrum pernix* (Luo, et al., 2007).

**3. Multiplicity of homologs** 

Olszewski, et al., 2008).

Shereda, et al., 2008). These SSB complexes occlude up to 65 and 35 nucleotides of ssDNA, respectively (Filipkowski & Kur, 2007; Meyer & Laine, 1990; Shereda, et al., 2008). Other, less common SSB configurations have also been identified, including an alternative homopentameric Deinococcal SSB (Norais, et al., 2009; Sugiman-Marangos & Junop, 2010). In general, the N-terminal region of the SSB contains the oligomerization domain while the C-terminal region is implicated in heterologous protein interactions (Shereda, et al., 2008).

Fig. 1. RPA and SSB structures from eukaryotes, bacteria, and archaea. (A) The canonical eukaryotic RPA comprises three protein subunits, each containing OB-folds designated DBD A-F. Yellow OB-folds are DNA interacting domains, blue domains are involved in heterologous protein interactions, and gray domains are involved in maintenance of RPA structure. The N-terminus of RPA2 is the primary phosphorylation domain, denoted by red Ps. (B) The canonical bacterial SSB functions as a homotetramer and contains a single OBfold (yellow) and a C-terminal heterologous protein interacting domain (blue) per monomer. (C) Archaeal RPAs are diverse and display characteristics of both eukaryotic RPA and bacterial SSB. Examples from *H. salinarum* are shown. RFA6 resembles bacterial SSB, whereas RFA1 is uniquely archaeal. The genes encoding RFA2 and RFA7, which are found in an operon, likely function as a multimeric protein, as do RFA3, RFA8, and RAL.

The eukaryotic RPA is a heterotrimer composed of a large RPA1 (~72 kDa), medium RPA2 (~32 kDa), and small RPA3 (~14 kDa) subunit. RPA1 and RPA2 tend to be conserved subunits with a variable RPA3, though considerable variation can occur throughout domains (Lin, et al., 2008; Wold, 1997). RPA1 contains four OB-folds referred to as DNA binding domains (DBD). DBD A and B are centrally located, DBD C is in the C-terminal region, and DBD F is in the N-terminal region. RPA2 contains a single centrally located OBfold, DBD D, and RPA3 a single centrally located OB-fold, DBD E (Binz, et al., 2004; Oakley, et al., 2009; Pretto, et al., 2010). ssDNA binding occurs primarily through interaction with DBD A-D, which together are capable of occluding approximately 30 nucleotides (Blackwell & Borowiec, 1994; Iftode, et al., 1999; C. Kim, et al., 1992; Theobald, et al., 2003). A binding polarity of the complex is achieved through decreased ssDNA affinity from DBD A to D (Oakley, et al., 2009; Pretto, et al., 2010). The N-terminal domain of RPA1 is implicated in heterologous protein interactions and regulation, particularly through DBD F, while the Cterminal domain is involved in heterotrimer structure interactions (Broderick, et al., 2010; Fanning, et al., 2006; Oakley, et al., 2009; Wold, 1997). The N-terminus of RPA2 contains a regulatory phosphorylation domain that affects interactions of RPA with other proteins, as well as its DNA binding kinetics (Machwe, et al., 2011; Nuss, et al., 2005; Oakley, et al., 2009; Patrick, et al., 2005; Vassin, et al., 2009). The C-terminal region of RPA2 is also involved in heterologous protein interactions and regulation. RPA3 consists almost entirely of DBD E and is thought to be involved primarily in heterotrimer formation, but potential roles in heterologous protein interactions have been identified (Cavero, et al., 2010; Wold, 1997).
