Zhiqiang Sun1, Yaqing Wang1, Mohtadin Hashemi1, Yuri L Lyubchenko2. 1. Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA. 2. Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA. Electronic address: ylyubchenko@unmc.edu.
Abstract
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 CC 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 CC 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.
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 CC 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 CC 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.
Authors: Basudeb Bhattacharyya; Nicholas P George; Tiffany M Thurmes; Ruobo Zhou; Niketa Jani; Sarah R Wessel; Steven J Sandler; Taekjip Ha; James L Keck Journal: Proc Natl Acad Sci U S A Date: 2013-12-30 Impact factor: 11.205
Authors: Maria Manosas; Senthil K Perumal; Piero R Bianco; Piero Bianco; Felix Ritort; Stephen J Benkovic; Vincent Croquette Journal: Nat Commun Date: 2013 Impact factor: 14.919