The Rad54 protein seed DNA repair synthesis
| Authors | |
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| Year of publication | 2009 |
| Type | R&D Presentation |
| MU Faculty or unit | |
| Citation | |
| Description | Rad54 is a key enzyme involved in the genome maintenance pathway of homologous recombination (HR). This protein has been implicated in several steps of HR, but its exact role(s) at each step is still not fully understood. We have identified a novel interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction can be abrogated by mutation of two hydrophobic residues in the conserved PCNA-interaction motif (PIP-box). This rad54 mutant (rad54-AA) is specifically defective in PCNA binding, retaining its ability to interact with Rad51, to promote in vitro strand exchange activity, shows similar ATPase activity to the wild-type protein, and is able to be recruited to HR foci in vivo. Despite this proficiency, rad54-AA mutant cells display a phenotype similar to the null in recombination assays and sensitivity to DNA damaging agents. Furthermore, the rad54-AA mutant shows a phenotype consistent with mutants that have defects in late HR steps, such as increased Rad52 focus duration and synthetic lethality with srs2 deletion. Since this mutant specifically affects interactions with the PCNA replication clamp, we tested the role of this mutant during the repair synthesis step of HR. Indeed, the rad54 PIP-box mutant displays defects in primer extension at the MAT locus. This is further supported by observation that Rad54 stimulates extension activity by Poldelta in vitro, likely mediated by PCNA interactions, since the rad54-AA mutant curtails this stimulation. We suggest that Rad54-PCNA interactions are critical during late steps of HR, and for allowing efficient DNA repair synthesis to proceed. |
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