The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin

Abstract

Significance In this work, we elucidate a checkpoint dampening mechanism in yeast. Using complementary biochemical and genetic approaches, we show that the Srs2 DNA helicase removes one of the first DNA damage sensors and the associated checkpoint kinase from chromatin, thus preventing hyperactivation of the DNA damage response. We further show that this role of Srs2 is separable from its well-known function as an antirecombinase and mainly accounts for Srs2’s contribution to genotoxin resistance. Our findings also shed light into potential means to regulate the dynamic association of RPA with single-stranded DNA in other cellular contexts and stimulate studies of checkpoint dampening and RPA regulation in other organisms. The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin in srs2 mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.