UvsW an orphan of SF2

T4 bacteriophage is a DNA virus that encodes 300 protein genes needed for various processes during the viral life cycle including the components of DNA replication, repair and recombination machineries. The replication of viral genome occurs by two separate mechanisms: origin-dependent replication during early stages of the viral life cycle and recombination-dependent replication (RDR) in the late stage. UvsW, one of the helicases encoded by the T4 genome, is associated with transition between these two modes of replication (Derr &

Kreuzer, 1990).

UvsW (UV sensitivity W) is a 58 kDa protein which together with other two proteins UvsX (strand invasion protein) and UvsY (mediator protein) was referred to as the WXY system, first shown to be involved in the tolerance of UV damage probably via recombinational repair pathway (Conkling & Drake, 1984).

The 503 aa long protein UvsW contains two domains that are separated by a short linker. The small N-terminal domain which comprises first 83 aa was shown to be structurally similar to dsDNA binding motif of the T4 transcription factor MotA and to the HARP domain of SMARCAL1 protein (see section 2.1.3.4) (Mason et al., 2014; Sickmier et al., 2004). The C-terminal domain is the SF2 helicase core containing two RecA domains (Kerr et al., 2007).

Initial experiments with UvsW-GST fusion proteins revealed ssDNA depen-dent ATPase activity of UvsW and its ability to unwind Y-shaped substrates composed of all-dsDNA arms, but inability to unwind linear dsDNA duplex (CarlesKinch et al., 1997). Further in vitro studies revealed wide spectra of activities including unwinding of DNA substrates such as partial and full replication forks, HJ, D-loop and RNA oligonucleotide containing R-loop, reversal of stalled replication forks and DNA strand branch migration (Dudas &

Kreuzer, 2001; Long & Kreuzer, 2009; Nelson & Benkovic, 2007; Webb et al., 2007). Additionally, UvsW is able to anneal complementary ssDNA in ATP dependent manner (Nelson & Benkovic, 2007).

The data obtained from biochemical and genetic studies of UvsW suggest that the enzyme has several roles in T4 bacteriophage life cycle including cation, repair and recombination. Transition between origin-dependent repli-cation and RDR late replirepli-cation is regulated by different pathways and involves UvsW dependent unwinding of R-loops that are vital for initiation of origin-dependent replication (Dudas & Kreuzer, 2001).

Recombination-dependent replication starts when a 3´ overhang of the repli-cated genome copy invades into homologous dsDNA and forms a D-loop which is further utilized by the viral replication machinery (Kreuzer & Brister, 2010).

Strand exchange process is mediated by viral recombination proteins UvsX, UvsY and UvsW. UvsW promotes the UvsX catalyzed strand exchange reaction and, thus, is directly involved RDR (Gajewski et al., 2011).

In addition, the branch migration activity of UvsW could also play a role in RDR via stabilization and migration of the formed HJ after strand exchange reaction (Webb et al., 2007). The RDR and DSB repair are tightly intercon-nected as DSB repair pathways are based on the same principles as RDR.

Therefore, the branch migration and strand exchange activities of UvsW could also be applied for DSB repair in the T4 genome. Interestingly, during SDSA pathway of DSB repair, the 3´ overhang of the inserted strand has to be slightly extended, removed from the D-loop and annealed to another complementary strand of DSB. The D-loop dissociation and ssDNA annealing activities of UvsW suggest that the protein is involved in aforementioned processes (Nelson

& Benkovic, 2007; Webb et al., 2007).

As previously noted, UvsW was shown to regress replication forks in vivo and in vitro (Long & Kreuzer, 2008, 2009). While regressed replication fork is an intermediate that could be processed in several ways, the biological role of this event needs further clarification. Nelson and Benkovic showed that when the T4 phage replication machinery encounters a lesion in the leading strand template it was capable to synthesize lagging strand further over the site of this

lesion before stalling (Figure 8A-C) (Nelson & Benkovic, 2010). Such a stalled replication fork with a gap in the leading strand is then reversed by UvsW and the lagging strand is used as the template to synthesize the leading strand (Figure 8D). Rewinding of the modified stalled fork by UvsW allows repli-cation to overcome the lesion and to continue the genome replirepli-cation (Figure 8E). In this process of the leading strand bypass, UvsW is able to switch randomly the strand and thereby catalyzes both the regression and restoration of the replication fork (Manosas et al., 2012).

The fact that bacteriophage genome encodes for single DNA polymerase incapable to bypass the lesion region (not acting as translesion polymerase) and no reports of any host polymerase participating in T4 replication cycle also supports the view that this lesion bypass mechanism is the main pathway in the phage life cycle (Manosas et al., 2012; Nelson & Benkovic, 2010).

Many helicases are regulated by their interaction partners and it seems likely that UvsW may also be regulated in a similar way. Therefore, further studies of potential interaction candidates such as the little-studied UvsW.1 together with the viral single stranded binding protein gp32 (Nelson & Benkovic, 2007), the recombination proteins UvsX and UvsY or some unknown protein could probably reveal new aspects of UvsW activities.

Figure 8. The lesion bypass mechanism in T4 bacteriophage. (A) Leading strand poly-merase encounters a lesion in the template. (B) The lagging strand could be synthesized further before the replication machinery stalls. (C) The stalled fork is then regressed and (D) the 5´overhang used as a template for synthesis. (E) The rewinded leading strand bypasses the lesion and replication can be restarted. Leading strand – blue, lagging stand – red, lesion – asterisk. Synthesized leading strand – purple.

2.2. Mitochondrial genome of the yeast