RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (−)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.

RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.

The RecG DNA helicase a key player in stalled replication fork rescue. The single-stranded DNA binding protein (SSB) participates in this process, but its role in the interaction of RecG with the fork remains unclear. We used atomic force microscopy (AFM) to visualize the interaction of RecG with a fork DNA in the presence of SSB. We discovered that SSB enhances RecG loading efficiency onto the DNA fork by threefold. Additionally, SSB interacts with RecG leading to the RecG remodeling. As a result, RecG separates from the fork, but remains bound to the DNA duplex. Moreover, in this new binding mode RecG is capable of translocation along the parental duplex DNA. We propose a model of RecG interaction with the replication fork involving two RecG binding modes. SSB plays the role of a remodeling factor defining the mode of RecG binding to the fork mediated by the SSB C-terminus. In the translocating mode, RecG remains in the vicinity of the fork and is capable of initiating the fork regression. Our results afford novel mechanistic insights into RecG interaction with the replication fork and provide the basis for further structural studies.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 M ammonium sulfate must be present, bringing into question the validity of these data as these are non-physiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using purification of tagged complexes, our results demonstrate that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the “storage form” of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB which, by virtue of its high affinity for single stranded DNA, allows these helicases to out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.