Interaction of T4 UvsW Helicase and Single-Stranded DNA Binding Protein gp32 through Its Carboxy-Terminal Acidic Tail

Abstract

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA binding protein, gp32, is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.

RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

“…5) or protein displacement (Supplementary Fig. S9)1746—two endergonic reactions. Despite their differences in structure and sequence homologies1819, UvsW and RecG present similar behaviour with processive and fast DNA-rewinding activities against large applied forces of up to 35 pN, with only a moderate reduction in the rate of rewinding.…”

“…5 and Supplementary Fig. S9)1746 prove that RecG and UvsW can efficiently use this work to unwind nascent strands or to displace bound proteins—processes requiring an energy input of few k B T (1–3  k B T ) for unwinding a single bp or several k B T for displacing DNA-binding proteins214748. The active mechanism for fork regression is in stark contrast to the passive or Brownian ratchet model proposed for hexameric branch-migration helicases37, such as RuvAB, or DnaB4950, in which the enzyme uses unidirectional motion to trap spontaneous bp fraying.…”

Interaction between phage BFK20 helicase gp41 and its host Brevibacterium flavum primase DnaG

“…Since gp41 is predicted to be an SF2 helicase and SSB proteins often bind to the single DNA strands produced after a helicase unwinds double-stranded DNA, this is likely to be a true interaction. Moreover, an interaction between a host SSB and a T4 phage helicase (gp32 and UvsW) has been previously reported (Perumal et al, 2013). Like BFK20 gp41, T4 UvsW also belong to the SF2 helicase family and has been shown to play an important role in recombination-dependent DNA replication and repair (Nelson and Benkovic, 2007).…”

Structure and mechanism of the phage T4 recombination mediator protein UvsY

“…6A), the gp32 proteins are arrayed such that the flexible C termini can engage the outer surface of the UvsY heptamer. Such an interaction would have to be relatively nonspecific to explain how the gp32 C terminus is able to recruit other T4 proteins such as UvsX, UvsW, Dda, and DNA polymerase (30,31). Shown in Fig.…”