Single-Molecule Study of RecG Activity at a 3-way DNA Junction

Abstract

In E. coli, replicative DNA polymerases are often stalled for various reasons: head-on collision of DNA polymerases, blockage by frozen DNA-protein complexes or DNA damage by UV and oxidative stress. In particular, DNA damages causing polymerase stalling should be properly repaired by DNA repair pathways such as NER and HR. In order to avoid the impasse due to polymerase stalling and repair the DNA damages eventually, the stalled fork (3-way junction) is regressed for a proper repair on the damaged site. In the process of fork regression, RecG is thought to play a pivotal role: at the back of the fork, it may pump DNA back in order to convert the 3-way junction to a 4-way chicken-foot structure whereby the damaged spot becomes rewound and prepared for repair process.In our study, we examined the activity of RecG at a 3-way junction at the single-molecule level. Using single-molecule FRET technique, we clarified how RecG unwinds newly-synthesized DNA strands and induces the conformational transition of branched DNA structures. We also investigated the effects of monovalent cation, ATP, and ATP analogs on RecG activity and measured the rate of unwinding of DNA by RecG.From our study, we found in detail how RecG functions: it binds to a 3-way junction resembling a stalled replication fork and unwinds the junction to a 4-way junction in an ATP-dependent manner.