The PCNA Interaction Protein Box in Rad54 is an Integral Part of Its ATPase Domain and is Required for Efficient DNA Repair and Recombination
Burgess, R.C., Sebesta, M., Sisakova, A., Marini, V.P., Lisby, M., Damborsky, J., Klein, H., Rothstein, R., Krejci, L.
PLOS ONE 8: e82630 (2013)
Rad54 is an ATP-driven translocase involved in the genome maintenance pathway ofhomologous recombination (HR). Although its activity has been implicated in several steps ofHR, its exact role(s) at each step are still not fully understood. We have identified a newinteraction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen(PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobicresidues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA).Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HRdefects similar to the null mutant, despite retaining its ability to interact with HR proteins and tobe recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might beimpaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AAmutant was defective in primer extension at the MAT locus as well as in vitro, but additionalbiochemical analysis revealed that this mutant also had diminished ATPase activity and aninability to promote D-loop formation. Further mutational analysis of the putative PIP-boxuncovered that other phenotypically relevant mutants in this domain also resulted in a loss ofATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, thisconserved domain is probably an extension of the ATPase domain III.