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Genomic analysis of replication profiles identifies RFCCtf18 as a key mediator of the replication stress response

ABSTRACT: Maintenance of genome stability during DNA replication depends on surveillance mechanisms that prevent fork collapse and regulate replication timing. To dissect the checkpoint pathways signalling paused forks, we have screened a collection of S. cerevisiae mutants for their ability to activate Rad53, using the repression of late origins as readout for checkpoint efficiency. These genomic data redefine the role of key checkpoint factors. Indeed, they show that DNA damage sensors such as the 9-1-1 clamp and its loader RFCRad24 are unexpectedly dispensable to signal paused forks. In contrast, we found that the alternative clamp loader RFCCtf18 is essential for the Mrc1-dependent activation of Rad53. Moreover, we report that forks collapse in ctf18delta mutants and activate a secondary checkpoint pathway that represses very late origins in a Rad9-dependent manner. We propose a novel and integrated view of the replication checkpoint in which different pathways cooperate to fine tune the cellular response to arrested forks.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE21014 | GEO | 2010/10/26

SECONDARY ACCESSION(S): PRJNA125757

REPOSITORIES: GEO

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Project description:In budding yeast, DNA lesions and stalled replication forks are sensed by the apical checkpoint kinase Mec1/ATR, which leads to the downstream activation of the effector kinase Rad53/CHK1. This activation depends on Rad9 and Mrc1, two checkpoint mediators that integrate the nature of the challenge in different phases of the cell cycle. Rad9 mediates the activation of the DNA damage checkpoint throughout the cell cycle, while the function of Mrc1 is restricted to the S phase of the cell cycle, when it travels with the replication fork and activates the DNA replication checkpoint in response to a variety of replication impediments. In this scenario, the role of Rad9 in S phase has been largely disregarded since the discovery of Mrc1, because Rad9 is dispensable for the timely activation of Rad53 in response to the drug hydroxyurea, which halts forks, and is only recruited to those stalled forks when Mrc1 is absent. Thus, Rad9 is simply believed to act as a backup pathway for Mrc1 during replication. We have re-evaluated the role of Rad9 when DNA damage arises during replication and characterized its functional interplay with Mrc1. To this end, we have used genome-wide approaches, single-molecule analysis, pulsed-field and 2D gel electrophoresis, as well as a careful combination of different replication-challenging drugs. We have found that both Mrc1 and Rad9 play distinct but complementary functions in the replication stress response during S phase, for they coordinate the early and late functions of Rad53, respectively. While Mrc1 is responsible for the fast activation of Rad53 in response to fork-halting drugs in order to repress late origins, Rad9 maintains Rad53 in an active state during prolonged fork arrest and is necessary to sustain this response for long periods. Remarkably, we also have found that Rad9 possesses the unprecedented activity of slowing down replication fork progression in response to DNA damage. This work thus restores the legitimate role of Rad9 as a central actor in the maintenance of genome integrity during replication. This has important implications for our understanding of the management of the checkpoint during perturbed replication in human cells, for Rad9 has three orthologues, 53BP1, BRCA1 and MDC1, whose contribution to this aspect of genome integrity remains largely unexplored.

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Genomic analysis of replication profiles identifies RFCCtf18 as a key mediator of the replication stress response

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