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

Abstract

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.

Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA

“…This core biochemical activity, which does not preclude other functions for HELB, could potentially promote many DNA transactions where RPA filaments are intermediates that might otherwise block downstream processing events. In this respect, HELB may share certain aspects of its function with the yeast Srs2 helicase ( 13 , 39 ). These findings have important implications for better understanding the biochemical basis for the roles that HELB might play in DNA repair and replication.…”

“…The filaments formed between RPA and ssDNA are critical intermediates in DNA replication, recombination, and repair ( 10 – 12 ). RPA not only shields ssDNA from nucleolytic degradation, but it is also involved in the recruitment or exclusion of other factors from ssDNA, the regulation of DNA replication and repair, and the initiation of cell signaling cues that link these pathways to the cell cycle and its progression through checkpoints ( 13 ). Interestingly, many helicases and helicase-like proteins share intimate physical and functional interactions with ssDNA binding proteins ( 14 , 15 ).…”

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments

“…The antirecombinase Srs2 is also involved during these early presynaptic complex assembly steps ( 1 – 4 ), a premise that is consistent with the presence of Srs2 in DNA damage-induced foci even when Rad51 is absent ( 80 ). Studies of Srs2 indicate that it readily removes RPA, Rad52, and Rad51 from ssDNA ( 17 – 20 , 31 – 33 ). Rad55–Rad57 helps to offset Rad51 removal by enhancing Rad51 rebinding, but it does not block Srs2 activity ( 36 ).…”

“…Specific protein–protein contacts between Srs2 and Rad51 help promote Rad51 removal by stimulating Rad51 ATP hydrolysis activity, leading to the formation of a Rad51–ADP complex, which has an inherently lower affinity for ssDNA compared to ATP-bound Rad51 ( 19 ). In addition to removing Rad51 from ssDNA, in vitro Srs2 can also readily remove ssDNA-bound RPA and Rad52, a well-studied recombination mediator involved in Rad51 presynaptic complex assembly, suggesting that Srs2 can act upon several ssDNA-binding proteins crucial for the early stages of HR ( 32 , 33 ).…”

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

“…This notion of two separable features of RPA is supported by our in vivo data, where we tested the cellular effects of deletion of Rtt105 along with three distinct DNA binding mutants of RPA. zm1 (K494A) and zm2 (N492D, K494R, K494R) mutants of RPA carry amino acid substitutions close to the Zn 2+ -finger binding domains in DBD-C and have reduced ssDNA binding activity (Dhingra et al, 2021). t33 is a well characterized RPA mutant with a S373P substitution in DBD-B (Chen et al, 1998; Deng et al, 2014).…”