Restriction of RecG translocation by DNA mispairing

Background: The RecG DNA helicase plays a crucial role in stalled replication fork rescue. We have recently discovered that interaction of RecG with single-strand DNA binding protein (SSB) remodels RecG, allowing it to spontaneously translocate upstream of the fork. Based on these findings, we hypothesized that mispairing of DNA could limit such translocation of RecG. Methods: Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. Results: We found that a C-C mispairing, at a distance of 30 bp from the fork position, prevents translocation of RecG over this mispairing. A G-bulge, placed at the same distance, also has a similar blocking efficiency. However, a C-C mispairing, 10 bp away from the fork, does not prevent RecG translocation beyond 10 bp distance, but decreases complex yield. Modeling of RecG-DNA complexes show that 10 bp distance from the fork is within the binding footprint of RecG on DNA. Conclusions: Our results suggest that the RecG translocation upstream of the replication fork is limited by mispairings in the parental arm of the replication fork. General significance These findings led us to propose dual functions for RecG, in which the thermally driven translocation of RecG can be a mechanism for the additional control of the DNA paring in which RecG can detect the lesions in front of the replication fork, adding to the fidelity of the DNA replication machinery.

Automated summary

The movement of RecG DNA helicase in DNA replication is limited by mispairs in the parental arm of the replication fork, providing an additional control mechanism for the DNA replication machinery.