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The interaction networks of the budding yeast and human DNA replication-initiation proteins.

ABSTRACT: DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G1 transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.

SUBMITTER: Wu R

PROVIDER: S-EPMC6464591 | biostudies-literature | 2019 Mar - Apr

REPOSITORIES: biostudies-literature

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The interaction networks of the budding yeast and human DNA replication-initiation proteins.

Wu Rentian R Amin Aftab A Wang Ziyi Z Huang Yining Y Man-Hei Cheung Marco M Yu Zhiling Z Yang Wei W Liang Chun C

Cell cycle (Georgetown, Tex.) 20190319 6-7

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G<sub>1</sub> transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin r ...[more]

PMID: 30890025

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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.

Dataset Information

The interaction networks of the budding yeast and human DNA replication-initiation proteins.

Publications

The interaction networks of the budding yeast and human DNA replication-initiation proteins.

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