MEC1-dependent phosphorylation of yeast RPA1 in vitro

Replication protein A (RPA) is a conserved single-stranded DNA (ssDNA) binding protein with well-characterized roles in DNA metabolism. RPA is phosphorylated in response to genotoxic stress and is required for efficient checkpoint function, although these aspects of RPA function are not well understood. We have investigated the association between RPA and the checkpoint kinase Mec1 in yeast. RPA and Mec1 were found to be physically associated during unperturbed cell growth and in response to DNA damage. Using a Mec1 immunoprecipitate (IP)-kinase assay, we show that the two large subunits, RPA1 and RPA2, are good substrates for Mec1 kinase. The major phosphorylation site of RPA1 was further investigated as it was found to be localized to its amino terminus (RPA1N), which is a non-ssDNA binding domain implicated in regulatory function. This phosphorylation site mapped to serine 178 and phosphorylation-defective mutant protein, expressed from rfa1-S178A, showed reduced physical interaction with Mec1. Phenotypic analysis in vivo revealed that the rfa1-S178A mutation affected the kinetics of RPA1 and Rad53 phosphorylation but did not otherwise affect the checkpoint response. We suggest that phosphorylation of RPA1N by Mec1 may function together with other checkpoint events to regulate the checkpoint response.