Processive and unidirectional translocation of monomeric UvsW helicase on single-stranded DNA.

DNA helicases couple the energy of ATP hydrolysis to the energetically unfavorable process of unwinding the DNA double helix (1). UvsW protein is one of three helicases from bacteriophage T4 and based on sequence identity is a member of the super family 2 (SF2) helicases (2). UvsW has several roles in T4 phage DNA replication and repair. UvsW governs the mode of replication initiation by unwinding the stably bound RNA transcripts (R-loops) from the T4 origins of replication (3). The removal of R-loops eliminates origin-dependent initiation, causing origin independent (recombinogenic) initiation to become the dominant mode of initiation (4). UvsW knockout mutations are sensitive to UV light or hydroxyurea, indicating a role in the processing of damaged DNA and stalled replication forks (5–7). In vitro, UvsW unwinds branched DNA substrates, suggesting that UvsW is responsible for the residual branch resolution activity in the absence of gp49 (8–10). In addition to branch migration activity, UvsW unwinds DNA substrates resembling recombination intermediates and stalled replication forks (11). UvsW unwinds D-loops that are formed from the ssDNA strand invasion into homologous dsDNA and is able to unwind both the leading and lagging strand arms of a model stalled replication fork (11). This led to the proposal that UvsW participates in both synthesis dependent strand annealing (SDSA) and replication fork regression DNA repair pathways (11). It was recently discovered that UvsW has strand annealing activity (11). Strand annealing activity is a common property among eukaryotic RecQ helicases such as Werner’s and Bloom’s syndrome helicases. It is thought that strand annealing is a physiologically important property, as only RecQ helicases with strand annealing activity are capable of catalyzing replication fork regression (12, 13). The annealing activity of UvsW does not require ATP but is increased in its presence. ATP hydrolysis accompanies the activation of ssDNA annealing since non-hydrolyzable ATPγS is an inhibitor rather than an activator of the reaction. The proposed mechanism for strand annealing in the absence of ATP involves the independent binding of two ssDNA strands at separate locations on UvsW, followed by spontaneous annealing of the properly aligned ssDNA strands (11, 14). To explain the ATP activation, it was proposed that ATP-driven translocation of UvsW along one of the two ssDNA strands is necessary in situations where annealing is prohibited due to non-complementarity or misalignment of the ssDNA. T4 ssDNA binding protein (gp32) attenuates strand annealing, as does UvsW.1. UvsW.1 is a small (9 kD) protein that physically associates with UvsW (11). A recent NMR structure of UvsW.1 revealed a highly electronegative four-helix bundle that resembles the HRDC domain from RecQ helicases (15). The precise role of UvsW.1 is not known, however based on analogy to the HRDC domain, it has been suggested that it may assist UvsW in substrate recognition (15). The structure of the ligand-free form of UvsW has been determined using X-ray crystallography (15, 16). The structure reveals two RecA-like motor domains, a variable domain at the N-terminal region and a small subdomain located near the C-terminus. The N-terminal variable domain resembles a “double wing” motif and contains a putative substrate binding site (17). However, the structure does not contain a DNA substrate or resemble other helicases outside of the RecA-like domains, making predictions regarding substrate recognition difficult. ATP is predicted to bind in the interface between the two RecA-like domains and a model for the ATP and ssDNA bound form has been generated (15). An inchworm model of DNA unwinding has been proposed for UvsW based on the similarity between the UvsW structure and the structures of other monomeric helicases, mainly the SF1 helicase PcrA (18). The inchworm model involves the repeated movements of the RecA-like motor domains that are driven by the hydrolysis and release of ATP and ADP + Pi, respectively. These movements are conformationally coupled to DNA translocation, which drive the helicase into the DNA duplex where it acts as a “wedge” to separate the DNA strands. The crystal structure of ligand-free form of UvsW is monomeric, however, the oligomeric state has not been investigated in solution. The mechanism of DNA unwinding is likely to be closely linked to the mechanism of DNA translocation (18). Indeed, most theoretical models of DNA unwinding are based primarily on the ability of the helicase to translocate on single-stranded (ss) or double-stranded (ds) DNA (19–21, 18). Consequently, a complete understanding of ssDNA translocation is necessary to accurately describe the mechanism of dsDNA unwinding at the molecular level. Here, we investigate the properties of UvsW translocation on an ssDNA lattice. We find that UvsW translocates on ssDNA uni-directionally and in an ATP-dependent fashion. UvsW is highly processive during ssDNA translocation and is able to resist inhibition by both an inactive mutant UvsW and a heparin trap. We also provide functional evidence indicating that UvsW is monomeric during ssDNA translocation. Together, these data represent a first step toward the mechanistic understanding of ssDNA translocation by UvsW.