Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein

Abstract

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 tale of SSB

“…Once this process was complete we removed the SH3 domain only, leaving the ligand in place to illustrate the potential binding location of the SSB IDL on RecG. Here, the ligand adopts a position on the wedge domain of RecG that is incompatible with fork binding and places the OB-folds of the SSB tetramer in a position to bind ssDNA, thereby facilitating the loading of RecG as shown previously (Sun et al, 2015). This interaction would also provide an explanation for SSB-mediated remodeling of the helicase, which prevents it from docking at a fork and instead, facilitating thermal sliding.…”

Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy

“…These issues were addressed in another study in which where AFM was used to characterize the interactions of fork DNA substrates with SSB and RecG. 62 …”

“…(C) and (F) are the distributions of the proteins distances from the left end for SSB (n=95) and RecG (n=37), respectively [reprinted with permission form Nature Publishing Group, Macmillan Publishers Ltd. Creative commons license, Copyright 2015]. 62 …”

“…The figure was reproduced from paper respectively [reprinted with permission form Nature Publishing Group, Macmillan Publishers Ltd. Creative commons license, Copyright 2015]. 62 …”

“…Model (i) shows the RecG position at the fork just after remodeling and (ii) shows the RecG position after translocation away from the fork respectively [reprinted with permission form Nature Publishing Group, Macmillan Publishers, Ltd. Creative commons license, Copyright 2015]. 62 …”