RESUMO
The DNA damage checkpoint is a highly conserved signaling pathway induced by genotoxin exposure or endogenous genome stress. It alters many cellular processes such as arresting the cell cycle progression and increasing DNA repair capacities. However, cells can downregulate the checkpoint after prolonged stress exposure to allow continued growth and alternative repair. Strategies that can dampen the DNA damage checkpoint are not well understood. Here, we report that budding yeast employs a pathway composed of the scaffold protein Rtt107, its binding partner Mms22, and an Mms22-associated ubiquitin ligase complex to downregulate the DNA damage checkpoint. Mechanistically, this pathway promotes the proteasomal degradation of a key checkpoint factor, Rad9. Furthermore, Rtt107 binding to Mms22 helps to enrich the ubiquitin ligase complex on chromatin and target the chromatin-bound form of Rad9. Finally, we provide evidence that the Rtt107-Mms22 axis operates in parallel with the Rtt107-Slx4 axis, which displaces Rad9 from chromatin. We thus propose that Rtt107 enables a bifurcated "anti-Rad9" strategy to optimally downregulate the DNA damage checkpoint.
RESUMO
Activation of the DNA damage checkpoint upon genotoxin treatment induces a multitude of cellular changes, such as cell cycle arrest, to cope with genome stress. After prolonged genotoxin treatment, the checkpoint can be downregulated to allow cell cycle and growth resumption. In yeast, downregulation of the DNA damage checkpoint requires the Srs2 DNA helicase, which removes the ssDNA binding complex RPA and the associated Mec1 checkpoint kinase from DNA, thus dampening Mec1 activation. However, it is unclear whether the 'anti-checkpoint' role of Srs2 is temporally and spatially regulated to both allow timely checkpoint termination and to prevent superfluous RPA removal. Here we address this question by examining regulatory elements of Srs2, including its phosphorylation, sumoylation, and protein-interaction sites. Our genetic analyses and checkpoint level assessment suggest that the RPA countering role of Srs2 is promoted by Srs2 binding to PCNA, which is known to recruit Srs2 to subsets of ssDNA regions. RPA antagonism is further fostered by Srs2 sumoylation, which we found depends on the Srs2-PCNA interaction. Srs2 sumoylation is additionally reliant on Mec1 and peaks after Mec1 activity reaches maximal levels. Collectively, our data provide evidence for a two-step model wherein checkpoint downregulation is facilitated by PCNA-mediated Srs2 recruitment to ssDNA-RPA filaments and the subsequent Srs2 sumoylation stimulated upon Mec1 hyperactivation. We propose that this mechanism allows Mec1 hyperactivation to trigger checkpoint recovery.
RESUMO
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.