Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

McClure, Allison W.; Diffley, John F.X.

doi:10.7554/elife.69726

Cited by 42 publications

(39 citation statements)

References 55 publications

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“…Hyper-phosphorylated Rad53 is undetectable in unperturbed mcm3-2KR cells (Fig 5D ), indicating that spontaneous DNA damage or replication stress is below the detection limit. Hydroxyurea (HU) treatment causes stalled DNA replication forks, which trigger Rad53 hyper-activation via the Mrc1 adaptor in the DNA replisome [37][38][39][40]. HU-induced (or Phleomycin-induced) Rad53 activation is dampened in mcm3-2KR cells compared to WT cells, consistent with the idea that Rad53 activation requires signaling via an intact DNA replisome, and that replisome levels are lowered in the mcm3-KR cells.…”

Section: Plos Geneticssupporting

confidence: 60%

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM

et al. 2022

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Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.

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Section: Plos Geneticssupporting

confidence: 60%

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM

et al. 2022

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“…[59,99] Other in vitro studies using a reconstituted replication system have elaborated that Mrc1 stimulates the activity of both the CMG helicase and Pol ε. [100,101] Further experiments using this system have shown that Rad53 phosphorylates Mrc1 and thereby suppresses its ability to stimulate DNA synthesis. Together, we propose that Rad53 phosphorylates Mrc1, which in turn slows down leading strand DNA synthesis under replication stress.…”

Section: Mechanistic Insights Into How Rad53 Couples Leading and Lagg...mentioning

confidence: 99%

“…Other studies have shown that Rad53 phosphorylates Mrc1, which may in turn inhibit the ability of Mrc1-Tof1-Csm3 to stimulate CMG helicase activity and potentially the activity of Pol ε as well. [8,88,100] Therefore, we hypothesize that Rad53 phosphorylates multiple replisome components to slow down DNA synthesis of leading and lagging strands simultaneously (Figure 2). Because the machinery involved in leading and lagging strand DNA synthesis function autonomously in vitro, we further suggest that these actions couple leading and lagging strand DNA synthesis and thereby prevent deleterious ssDNA template exposure under replication stress.…”

Section: Mechanistic Insights Into How Rad53 Couples Leading and Lagg...mentioning

confidence: 99%

Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress

Zhang

2022

BioEssays

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DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU‐IP‐ssSeq, strand‐specific sequencing technologies that permit analysis of protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1‐Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA.

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“…It has been shown that the budding yeast ortholog of the checkpoint adaptor protein Claspin, together with the Timeless:Tipin orthologs, is required to maintain coupling between the CMG helicase and the replisome [ 79 , 80 , 81 ]. Both budding yeast and human Claspin are required to regulate replication fork speed [ 82 , 83 , 84 ].…”

Section: Beyond Replication Initiation: the Role Of Ddk In Replicatio...mentioning

confidence: 99%

DDK: The Outsourced Kinase of Chromosome Maintenance

Gillespie

Blow

2022

Biology

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The maintenance of genomic stability during the mitotic cell-cycle not only demands that the DNA is duplicated and repaired with high fidelity, but that following DNA replication the chromatin composition is perpetuated and that the duplicated chromatids remain tethered until their anaphase segregation. The coordination of these processes during S phase is achieved by both cyclin-dependent kinase, CDK, and Dbf4-dependent kinase, DDK. CDK orchestrates the activation of DDK at the G1-to-S transition, acting as the ‘global’ regulator of S phase and cell-cycle progression, whilst ‘local’ control of the initiation of DNA replication and repair and their coordination with the re-formation of local chromatin environments and the establishment of chromatid cohesion are delegated to DDK. Here, we discuss the regulation and the multiple roles of DDK in ensuring chromosome maintenance. Regulation of replication initiation by DDK has long been known to involve phosphorylation of MCM2-7 subunits, but more recent results have indicated that Treslin:MTBP might also be important substrates. Molecular mechanisms by which DDK regulates replisome stability and replicated chromatid cohesion are less well understood, though important new insights have been reported recently. We discuss how the ‘outsourcing’ of activities required for chromosome maintenance to DDK allows CDK to maintain outright control of S phase progression and the cell-cycle phase transitions whilst permitting ongoing chromatin replication and cohesion establishment to be completed and achieved faithfully.

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Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

Cited by 42 publications

References 55 publications

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM

Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress

DDK: The Outsourced Kinase of Chromosome Maintenance

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