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H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly

ABSTRACT: The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE61030 | GEO | 2014/09/12

SECONDARY ACCESSION(S): PRJNA260114

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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H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly

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