**1. Introduction**

430 DNA Repair

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#### **1.1 Recombinational repair**

*H*omologous *r*ecombination (HR) is essential for genetic diversity and genome stability. The conserved RecA-like recombinases promote pairing and consequent exchange of fragments between two homologous DNA molecules during conjugation in bacteria and meiotic recombination in eukaryotes. HR is a main DNA repair pathway particularly important in case of large-scale DNA damages, including chromosome or *d*ouble-*s*tranded (ds) *D*NA *b*reaks (DSBs) and long *s*ingle-*s*tranded (ss) DNA *g*aps (SSGs) (Cox, 1991; Kowalczykowski et al., 1994). The broken chain is paired with the intact DNA, which serves as a template for the synthesis of the damaged DNA. The same recombinases are also involved in the repair and origin-independent restart of stalled DNA replication, a frequently occurring event in every cell (Cox et al., 2000; Kowalczykowski, 2000; Kuzminov, 2001).

HR is initiated by the cooperative binding of RecA recombinase to ssDNA hundreds or thousands nucleotides long forming nucleoprotein filament, a so called presynaptic complex often designated as RecA\*. The presynaptic complex can bind homologous dsDNA and exchange a DNA strands. RecA\* has multiple activities beyond the strand invasion and exchange (Figure 1). Those include triggering DNA damage SOS response through stimulation of LexA autocleavage (Rehrauer et al., 1996) and activation of UmuD subunit of the error-prone DNA polymerase PolV important for translesion synthesis to bypass small-scale DNA errors (Jiang et al., 2009; Rajagopalan et al., 1992). RecA\* was also suggested to stabilize and maintain stalled replication fork during DNA repair (Courcelle et al., 1997). Consequently, RecA binding to DNA is regulated at multiple levels (Cox, 2007).

#### **1.2 Recombination mediator proteins**

Transient ssDNA regions generated during replication are protected by ssDNA binding proteins like bacterial *ss*DNA *b*inding (SSB) protein and eukaryotic *r*eplication *p*rotein *A* (RPA), which prevent recombinase binding. Under DNA damage conditions, ubiquitous *r*ecombination *m*ediator *p*roteins (RMPs) overcome inhibitory effect of SSB and initiate presynaptic complex formation (Fig. 1)(Beernink and Morrical, 1999; Symington, 2002). RMPs are not directly involved in the repair of specific DNA damages, but they regulate initiation of multiple DNA repair pathways and damage response signaling cascades (Courcelle, 2005; Kowalczykowski, 2005; Lee and Paull, 2005; Moynahan et al., 2001;

ATP-Binding Cassette Properties of Recombination Mediator Protein RecF 433

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;

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

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

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

Kowalczykowski, 2005; Mohaghegh and Hickson, 2001; Yang et al., 2005).

replication restart at the site of disruption.

**2.2 Mechanism of RecOR activities**

al., 2006).

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

Fig. 1. The ssDNA hand-off from ssDNA binding proteins to RecA-like recombinase triggers multiple DNA damage response pathways important for DNA repair and originindependent restart of stalled replication. Such DNA transaction is regulated by RMPs.

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 controversial issues.
