Article

Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Department of Genetics, Harvard University Medical School, Boston, MA 02115, USA.
Molecular cell (Impact Factor: 14.46). 06/2012; 47(3):396-409. DOI: 10.1016/j.molcel.2012.05.024
Source: PubMed

ABSTRACT Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.

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    • "In response to replication blocks, PCNA undergoes monoubiquitination that recruits translesion polymerases and stimulates direct replication bypass. Additionally , PCNA can become polyubiquitinated with K63-linked chains to promote recombination-dependent DNA synthesis across DNA lesions by template-switching mechanisms and recruits ZRANB3 to sites of replication stress (Ciccia et al., 2012). FANCI and FANCD2 respond to DNA interstrand crosslinks that arrest replication forks and are recruited to damaged DNA where they are ubiquitinated and promote repair. "
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    • "We also commonly observed that when the two halves of the Holliday and replication fork junctions were not pre-annealed, hsHARP and dmHARP increased the annealing rate of the two halves during the final incubation (Figure S3). To test whether dmHARP was competent to disrupt D-loops as previously shown for hsHARP [10], a 90 bp D-loop was formed on a supercoiled plasmid with RecA and RPA and subsequently purified (Figure 2B, lane 1). Both hsHARP and dmHARP catalyzed D-loop disruption (lanes 2-4), but the reactions with dmHARP were less complete within the time frame of this assay. "
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