Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB Journal Article
| Authors: | Gupta, S.; Yeeles, J. T. P.; Marians, K. J. |
| Article Title: | Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB |
| Abstract: | 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.Wehave 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.Wefind 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. |
| Keywords: | replication fork |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 289 |
| Issue: | 41 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2014-10-10 |
| Start Page: | 28388 |
| End Page: | 28398 |
| Language: | English |
| DOI: | 10.1074/jbc.M114.587907 |
| PROVIDER: | scopus |
| PMCID: | PMC4192491 |
| PUBMED: | 25138217 |
| DOI/URL: | |
| Notes: | Export Date: 3 November 2014 -- Source: Scopus |
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