RecG Interaction with the DNA Replication Fork. The Role of E. coli SSB Protein

Abstract

The RecG DNA helicase is a guardian of the bacterial genome. It binds to a variety of forked DNA structures thereby minimizing pathological DNA replication and facilitating stalled replication fork rescue. It is becomingly increasingly clear that SSB plays a critical role in the function of RecG, but the mechanisms of its interaction with SSB remain unclear. Here we use the atomic force microscope (AFM) to image the structure of RecG with a model fork substrate in the presence or absence of the single strand DNA binding protein (SSB). The DNA substrate has a 3′-end 69nt single stranded DNA (ssDNA) segment inserted between the two DNA duplexes. This design mimics a stalled fork with an ssDNA gap in the leading strand. The SSB proteins bind very specifically to the ssDNA segment with a yield of ∼90%. RecG protein also binds to the fork but the yield was ∼10%. However, the RecG loading on the same substrate increases three-fold in the presence of SSB suggesting that SSB facilitates RecG loading onto the fork. Moreover, in the presence of SSB, RecG becomes capable of translocating along the DNA duplex in an ATP hydrolysis-independent manner. No such mobility of RecG was observed in the absence of SSB. The preferable translocation direction is moving of RecG along parental arm which implies RecG could clear obstacles bound ahead of the fork. This novel activity of SSB requires the SSB C-terminus as a truncated SSB mutant does not substitute for wild type. SSB loading of RecG and subsequent translocation are unaffected by ATP, ADP or the non-hydrolyzable analog ATP-γ-S. Overall the results obtained reveal novel properties of RecG and highlight a new chaperone-type role of SSB in the DNA repair process.