**2. RecFOR recombination mediators**

#### **2.1 RecF pathway**

The RecF was discovered as an alternative to RecBC pathway in genetic screens based on frequency of conjugation recombination in *E. coli*, and was found to be important for postreplication repair of extended SSGs (Horii and Clark, 1973; Lovett and Clark, 1983; Wang and Smith, 1984). Later, it was shown that *recF* mutants are even more hypersensitive to UV radiation than *RecBC,* that RecF pathway plays a major role in replication restart under UV damage conditions, and that RecF is involved in DSBs repair in the absence of RecBC and SbcBC (Clark, 1991; Courcelle, 2005; Courcelle et al., 1997; Ivancic-Bace et al., 2003; Kidane et al., 2004; Kusano et al., 1989; Whitby and Lloyd, 1995; Zahradka et al., 2006). Sequencing of new genomes revealed the ubiquitous nature of RecF pathway proteins found in most bacteria (Rocha et al., 2005), including the radiation resistant bacteria *Deinococcus radiodurans* (Bentchikou et al., 2010; Cox et al., 2010; Chang et al., 2010; Makarova et al.,

Williams et al., 2007). In addition to presynaptic complex formation, many RMPs also promote DNA annealing (Luisi-DeLuca and Kolodner, 1994; Sugiyama et al., 1998). The importance of RMPs is reflected by the fact that recombination and repair pathways are often named after specific RMPs, e. g. RecF, RecBC, Rad52 pathways. RMPs include phage UvsY (Sweezy and Morrical, 1999), prokaryotic RecBCD and RecFOR proteins (Fujii et al., 2006; Kolodner et al., 1985; Lloyd and Thomas, 1983; Wang and Smith, 1983), and numerous eukaryotic members (Symington, 2002). Mutations of human RMPs are associated with cancer predisposition, mental retardation, UV-sensitivity and premature aging (Ouyang et

Fig. 1. The ssDNA hand-off from ssDNA binding proteins to RecA-like recombinase triggers

While ssDNA-binding proteins and RecA-like recombinases are well characterized, the mechanism of RMPs function remains poorly understood. Recent studies revealed a variety of RMPs structural domains. (Koroleva et al., 2007; Lee et al., 2004; Leiros et al., 2005; Makharashvili et al., 2004; Singleton et al., 2002; Yang et al., 2002). The diversity of RMPs structural domains reflects the plethora of different DNA damage response scenarios regulated by these proteins. The focus of this review is prokaryotic RMP RecF. Although a major bacterial recombination repair pathway is named after RecF, the mechanism of RecF activity and even its functional role remains one of the least understood and most

The RecF was discovered as an alternative to RecBC pathway in genetic screens based on frequency of conjugation recombination in *E. coli*, and was found to be important for postreplication repair of extended SSGs (Horii and Clark, 1973; Lovett and Clark, 1983; Wang and Smith, 1984). Later, it was shown that *recF* mutants are even more hypersensitive to UV radiation than *RecBC,* that RecF pathway plays a major role in replication restart under UV damage conditions, and that RecF is involved in DSBs repair in the absence of RecBC and SbcBC (Clark, 1991; Courcelle, 2005; Courcelle et al., 1997; Ivancic-Bace et al., 2003; Kidane et al., 2004; Kusano et al., 1989; Whitby and Lloyd, 1995; Zahradka et al., 2006). Sequencing of new genomes revealed the ubiquitous nature of RecF pathway proteins found in most bacteria (Rocha et al., 2005), including the radiation resistant bacteria *Deinococcus radiodurans* (Bentchikou et al., 2010; Cox et al., 2010; Chang et al., 2010; Makarova et al.,

multiple DNA damage response pathways important for DNA repair and originindependent restart of stalled replication. Such DNA transaction is regulated by RMPs.

controversial issues.

**2.1 RecF pathway** 

**2. RecFOR recombination mediators** 

al., 2008; Powell et al., 2002; Tal et al., 2009; Thompson and Schild, 2002).

2001). RecF forms an epistatic group with RecO and RecR proteins (Asai and Kogoma, 1994; Courcelle et al., 1997; Courcelle and Hanawalt, 2003; Horii and Clark, 1973; Kolodner et al., 1985; Wang and Smith, 1984). All three proteins are equally important for recombinational repair in most genetic screens, although they do not form triple complex in solution. RecF and RecR genes are often located in DNA replication operons on chromosome, with the exception of extremophiles like *T. thermophiles* and *D. radiodurans* (Ream and Clark, 1983; Ream et al., 1980). In *E. coli*, RecF is co-transcribed with major subunits of replication machinery, DnaA and DnaN (Perez-Roger et al., 1991; Villarroya et al., 1998). RecF pathway proteins share either sequence or structural homology or functional similarities with eukaryotic proteins such as WRN, BLM, RAD52, and BRCA2, which are associated with cancer predisposition and premature aging when mutated (Karow et al., 2000; Kowalczykowski, 2005; Mohaghegh and Hickson, 2001; Yang et al., 2005).

Genetic studies demonstrated that RecF regulates several DNA repair and recombination pathways but is not directly involved in repair of specific DNA damage. For example, in R*ecF* mutants DNA lesions are removed with the efficiencies comparable to wild-type cells, while the UV resistance is strongly compromised (Courcelle et al., 1999; Rothman and Clark, 1977). RecF-mediated loading of RecA on ssDNA is required for the maintenance of arrested replication forks, fot the protection and processing of DNA ends to permit DNA repair and replication restart at the site of disruption.

The regulatory role of RecF in replication restart is further supported by examples where RecF impairs cell survival, like in thymine starvation experiments (Nakayama et al., 1982). Another example is revealed by genetic studies of DNA helicases UvrD and Rep (Petit and Ehrlich, 2002). Mutants lacking both helicases are not viable and *RecF* mutations suppress the lethality of the E. coli *Rep/UvrD* double mutant. UvrD helicase disassembles RecA\* filaments, the reaction opposite to that of RecFOR, while Rep helicase promotes replication through transcription sites (Boubakri et al., 2010; Centore and Sandler, 2007; Heller and Marians, 2005; Lane and Denhardt, 1975; Veaute et al., 2005). The frequent pausing of the replication fork can potentially stimulate RecF-mediated initiation of RecA\* filament formation leading to illegitimate recombination in the absence of UvrD (Mahdi et al., 2006).

#### **2.2 Mechanism of RecOR activities**

The involvement of all three RecF, -O and -R proteins in HR initiation is well documented by genetic studies. However, the mechanism of their activities in the initiation process remains poorly understood, particularly with respect to RecF. RecO and RecR alone are sufficient to promote formation of the RecA filament on SSB-bound ssDNA (Cox, 2007; Umezu et al., 1993). RecO binds DNA and the C-terminal tail of SSB and these interactions are critical for RecOR function, at least in the absence of RecF (Inoue et al., 2011; Manfredi et al., 2010; Ryzhikov et al., 2011; Sakai and Cox, 2009; Umezu and Kolodner, 1994). In addition, RecO anneals complimentary ssDNA strands protected by cognate SSB (Kantake et al., 2002; Luisi-DeLuca and Kolodner, 1994), resembling the properties of the eukaryotic RMPs, Rad52 and BRCA2 (Grimme et al., 2010; Mazloum et al., 2007; Sugiyama et al., 1998). RecR binds either RecO or RecF (Makharashvili et al., 2009; Umezu and Kolodner, 1994; Webb et al., 1995, 1997). Although *E. coli* RecR does not bind DNA at submillimolar concentrations, it significantly affects DNA binding properties of both RecO and RecF (Kantake et al., 2002; Makharashvili et al., 2009; Webb et al., 1999). RecR inhibits DNA annealing properties of RecO, even though RecOR complex binds both ss- and dsDNA. In

junction.

reactions.

**3. Structural studies of RecF** 

**3.1 RecF is an ABC ATPase** 

ATP-Binding Cassette Properties of Recombination Mediator Protein RecF 435

evidence of direct involvement of RecF in the initiation of RecA\* filament formation was recently demonstrated in experiments with the SSB mutant lacking conserved C-terminus peptide. This SSB mutant inhibits RecOR-mediated recombination initiation, likely due to lack of interaction of SSB with RecO (Sakai and Cox, 2009). Surprisingly, RecF rescues the RecOR function with this SSB mutant, even on ssDNA plasmids without ds/ssDNA

The amino acid sequence of RecF contains three conserved motifs characteristic of ATPbinding cassette (ABC) ATPases: Walker A, Walker B, and a "signature" motif. Walker A, or P-loop, is a nucleotide binding site found in a variety of ATPases (Walker et al., 1982). Walker B motif provides acidic amino acids important for coordination of a water molecule and a metal ion during the hydrolysis of a triphosphate nucleotide bound to the Walker A motif. The signature motif is a unique feature of ABC ATPases, a diverse family of proteins ranging from membrane transporters to DNA-binding proteins (review in (Hopfner and Tainer, 2003). ATP-dependent dimerization is a common feature of this class of proteins. Signature motif residues interact with the nucleotide bound to an opposite monomer (Hopfner et al., 2000). This motif is important for both ATP-dependent dimerization and subsequent ATP hydrolysis. ABC ATPases are not motor proteins and utilize ATP binding and hydrolysis as a switch or sensor mechanism, regulating diverse signaling pathways and

DNA-binding ABC ATPases include DNA mismatch and nucleotide excision repair enzymes (Ban and Yang, 1998; Junop et al., 2001; Obmolova et al., 2000; Tessmer et al., 2008), structural maintenance of chromosome (SMC) proteins cohesin and condensin (Strunnikov, 1998), and DSBs repair enzyme Rad50 (Hirano et al., 1995). SMCs and Rad50 are characterized by the presence of a long coiled-coil structural domain inserted between Nand C-terminal halves of the globular head domain (Haering et al., 2002). RecF lacks a coiled-coil region, but it does exhibit an ATP-dependent DNA binding and a slow DNAdependent ATP hydrolysis activity (Hegde et al., 1996; Madiraju and Clark, 1992; Webb et al., 1995). However, the SMC-like properties of RecF and their role in recombinational repair have not been addressed. Previously, only Walker A motif has been shown to be critical for RecF function (Sandler et al., 1992; Webb et al., 1999). All known ABC-type ATPases function as a heterooligomeric complexes in which a sequence of inter- and intramolecular interactions is triggered by the ATP-dependent dimerization and the dimerdependent ATP hydrolysis (Deardorff et al., 2007; Dorsett, 2011; Hopfner and Tainer, 2003; Junop et al., 2001; Moncalian et al., 2004; Smith et al., 2002). Thus, RecF may function in recombination initiation through a multistep pathway of protein-protein and DNA-protein

The diversity of ABC ATPases makes it difficult to predict to which subfamily RecF belongs to based on sequence comparison. RecF is a globular protein lacking long coiled-coil domains of Rad50 and SMC proteins. However, it does not have significant sequence similarity beyond three major motifs with globular DNA binding proteins like MutS. We crystalized and solved a high resolution structure of RecF from *D. radiodurans* (DrRecF) (Fig.

interactions regulated by ATP-dependent RecF dimerization.

**3.2 Structural similarity of RecF with Rad50 head domain**

addition to initial loading of RecA, RecOR further stimulate homologous recombination by preventing the dissociation of RecA\* filament from ssDNA in *E. coli* (Bork et al., 2001). Somewhat different properties were reported for *Bacillus subtilis* RecO, which does not require RecR for initiation of RecA\* formation (Manfredi et al., 2008; Manfredi et al., 2010). Crystal structures of all three proteins and of the RecOR complex from *D. radiodurans* have been reported (Koroleva et al., 2007; Lee et al., 2004; Leiros et al., 2005; Makharashvili et al., 2004; Timmins et al., 2007). RecR structure resembles that of a DNA clamp-like tetramer (Lee et al., 2004). However, the role of a potential DNA clamp in RMPs-mediated reaction is unknown. Moreover, in the crystal structure of RecOR complex RecO occupies large portion of the clamp inner space. Such conformation makes it challenging to predict functionally relevant interaction of the complex with DNA. Another intriguing fact is that the crystal structure of RecO did not resemble any structural features of its functional eukaryotic analog Rad52 (Leiros et al., 2005; Makharashvili et al., 2004; Singleton et al., 2002), which supports two identical reactions.

#### **2.3 Ambiguities of RecF function**

In contrast to genetic data, initial biochemical studies did not reveal the function of RecF in recombination initiation (Umezu et al., 1993). RecF binds both ss- and dsDNA in the presence of ATP, and it is a weak DNA-dependent ATPase (Griffin and Kolodner, 1990; Madiraju and Clark, 1991, 1992). It interacts with RecR in the presence of ATP and DNA (Webb et al., 1999). Surprisingly however, RecF was initially shown to play an inhibitory role during RecOR-mediated loading of RecA on SSB-protected ssDNA (Umezu et al., 1993). The UV-sensitivity of *RecF* mutant can be suppressed by RecOR overexpression, suggesting that RecF plays a regulatory role (Sandler and Clark, 1994). In agreement with this hypothesis, RecF dramatically increases the efficiency of RecOR-mediated RecA loading at ds/ssDNA junctions with a 3' ssDNA extension under specific conditions (Morimatsu and Kowalczykowski, 2003). RecF was suggested to recognize specific DNA junction structure to direct RecA loading at the boundary of SSGs. While initial experiments demonstrated such a preference (Hegde et al., 1996), later work did not support the binding preference of RecF to DNA junction (Webb et al., 1999). Purified RecF tends to gradually aggregate in solution (Webb et al., 1999). Apparently, nonspecific high molecular weight RecF aggregates interact with DNA resulting in the inhibitory effect of RecF or false positive interactions of RecF with specific DNA substrates (Hegde et al., 1996). In addition, RecFR complex limits the extension of RecA\* beyond SSGs, the observation indirectly supporting RecF specificity towards boundaries of SSGs while in complex with other proteins (Webb et al., 1997).

RecF is co-transcribed with the replication initiation protein DnaA and with the β-clamp subunit of DNA polymerase III DnaN. However, its open reading frame is usually shifted by one or two nucleotides relatively to that of DnaN (Villarroya et al., 1998). *E. coli RecF* gene also has multiple rear codons. Thus, expression of RecF is likely to be down regulated at translational level. Consequently, there are only a few copies of RecF in an *E. coli* cell.

How RecF promotes recombination remains an open question. The ability of RecFR complex to limit extension of RecA\* filament beyond the SSGs suggests that the RecFR complex may specifically interact with RecA\*. However, no direct observation of such interactions has been reported so far. RecF also binds RecX protein (Lusetti et al., 2006). RecX is a negative regulator of presynaptic complex formation, which inhibits filament extension by binding to RecA. RecF scavenges RecX from solution through direct interaction, thus diminishing negative regulatory effect of RecX (Drees et al., 2004; Lusetti et al., 2006). Additional evidence of direct involvement of RecF in the initiation of RecA\* filament formation was recently demonstrated in experiments with the SSB mutant lacking conserved C-terminus peptide. This SSB mutant inhibits RecOR-mediated recombination initiation, likely due to lack of interaction of SSB with RecO (Sakai and Cox, 2009). Surprisingly, RecF rescues the RecOR function with this SSB mutant, even on ssDNA plasmids without ds/ssDNA junction.
