Regression of Replication Forks Stalled by Leading-strand Template Damage II. Regression by RecA is Inhibited by SSB [DNA and Chromosomes]

August 19th, 2014 by Gupta, S., Yeeles, J. T. P., Marians, K. J.

Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that RecA is unable to regress the stalled fork in the presence of the replisome and SSB. If the replication proteins are removed from the stalled fork, RecA will catalyze net regression as long as the Okazaki fragments are sealed. RecA-generated Holliday junctions can be detected by RuvC cleavage, although this is not a robust reaction. On the other hand, extensive branch migration by RecA, where a completely unwound product consisting of the paired nascent leading and lagging strands is produced, is observed under conditions where RuvC activity is suppressed. This branch migration reaction is inhibited by SSB, possibly accounting for the failure of RecA to generate products in the presence of the replication proteins. Interestingly, we find that the RecA-RuvC reaction is supported to differing extents depending on the template damage: Templates carrying a cyclopyrimidine dimer elicit more RecA-RuvC product than those carrying a synthetic abasic site. This difference could be ascribed to a higher affinity of RecA binding to DNAs carrying a thymidine dimer than to those with an abasic site.

Regression of Replication Forks Stalled by Leading-strand Template Damage I. Both RecG and RuvAB Catalyze Regression, but RuvC Cleaves the Holliday Junctions Formed by RecG Preferentially [DNA and Chromosomes]

August 19th, 2014 by Gupta, S., Yeeles, J. T. P., Marians, K. J.

The orderly progression of replication forks formed at the origin of replication in Escherichia coli is challenged by encounters with template damage, slow moving RNA polymerases, and frozen DNA-protein complexes that stall the fork. These stalled forks are foci for genomic instability and must be reactivated. Many models of replication fork reactivation invoke nascent strand regression as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecG and RuvAB, two proteins commonly thought to be involved in the process, using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that both RecG and RuvAB can regress the stalled fork in the presence of the replisome and SSB; however, RuvAB generates a completely unwound product consisting of the paired nascent leading and lagging strands, whereas RuvC cleaves the Holliday junction generated by RecG-catalyzed fork regression. We also find that RecG stimulates RuvAB-catalyzed regression, presumably because it is more efficient at generating the initial Holliday junction from the stalled fork.
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