RPA in Homologous Recombination


RPA and the BRCA1-BRCA2 Pathway

When cells encounter DNA damage in the form of double-strand breaks (DSBs), once the cell is committed to end resection, the binding of replication protein A (RPA) to the resected end is the next key step. RPA binding sets off a chain of events that result in the recruitment of HR repair proteins (including BRCA1 and BRCA2) to the sites of DNA damage. RPA, a heterotrimeric protein, becomes hyper-phosphorylated on the N-terminal end of its second subunit (RPA2). Hyper-phosphorylation of RPA2 is necessary for HR and only phospho-RPA2 shows co-localization with Rad51.

Our goal is to understand the regulation and kinetics of protein-protein interactions at sites of DNA damage. We are investigating whether hyper-phosphorylation of RPA2 mediates the interaction with, and the recruitment of, other HR proteins. We are using wild-type and mutant forms of RPA2 that are not capable of being phosphorylated to discover novel protein-protein interactions utilized in HR repair. Fluorescently tagged proteins have been made to study how these proteins get recruited to DNA damage sites. An understanding of the protein-protein interactions mediated by hyper-phosphorylated RPA2 will provide insight into how the DNA repair machinery is engaged in homologous recombination. We already know that BRCA1 is a determinant of RPA2 phosphorylation, and BRCA2 has no effect on this initiating step. A complex web of protein-protein interactions is involved in linking RPA modifications to BRCA-pathway engagement.

RPA and p53-Mediated Suppression of Homologous Recombination

p53 plays a key role in the regulation of homologous recombination (HR) during DNA replication. Recent studies have shown that human p53 controls HR events through its interaction with the RPA protein, since specific p53 mutations (D48/D49H and W53S/F54S) specifically disrupt RPA binding and fail to suppress HR, but maintain checkpoint and apoptotic function of p53.

We have recently found that the corresponding mutation in mice (p53-F50S/F51S) also abolishes the ability of p53 to control HR after replication block. Furthermore, F50S/F51S mutation increases the number of Rad51 foci compared to WT p53 in the presence of hydroxyurea (replication block), but not in irradiated cells. Interestingly, the p53 F50Q/F51S mutation has only a minor effect on the interaction with RPA, and this p53 mutant retains most of the suppressive effect on HR. A knock-in mouse containing the F50S/F51S mutation has been generated and has the cellular phenotype described. In vivo checkpoint and apoptotic activity is maintained, and the mouse colony is under observation for tumor development.