MDC1 collaborates with TopBP1 in DNA replication checkpoint control

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1–MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.

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Faculty Opinions recommendation of The DNA replication checkpoint response stabilizes stalled replication forks.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1000352.6760 ◽

2001 ◽

Author(s):

Antony Carr

Keyword(s):

Dna Replication ◽

Replication Forks ◽

Replication Checkpoint ◽

Checkpoint Response ◽

Dna Replication Checkpoint ◽

Stalled Replication Forks

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Overlapping Roles of the Spindle Assembly and DNA Damage Checkpoints in the Cell-Cycle Response to Altered Chromosomes in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/161.2.521 ◽

2002 ◽

Vol 161 (2) ◽

pp. 521-534

Author(s):

Peter M Garber ◽

Jasper Rine

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Spindle Checkpoint ◽

Dna Lesions ◽

Replication Proteins ◽

Replication Checkpoint ◽

Dna Damage Checkpoints ◽

Dna Replication Checkpoint ◽

Mcm Proteins ◽

Stalled Replication Forks

Abstract The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.

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DNA Replication Checkpoint Control Mediated by the Spindle Checkpoint Protein Mad2p in Fission Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m403231200 ◽

2004 ◽

Vol 279 (45) ◽

pp. 47372-47378 ◽

Cited By ~ 22

Author(s):

Izumi Sugimoto ◽

Hiroshi Murakami ◽

Yuko Tonami ◽

Akihiko Moriyama ◽

Makoto Nakanishi

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Spindle Checkpoint ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Checkpoint Protein ◽

Dna Replication Checkpoint

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Cdc18p can block mitosis by two independent mechanisms

Journal of Cell Science ◽

10.1242/jcs.111.20.3101 ◽

1998 ◽

Vol 111 (20) ◽

pp. 3101-3108 ◽

Cited By ~ 2

Author(s):

E. Greenwood ◽

H. Nishitani ◽

P. Nurse

Keyword(s):

Dna Replication ◽

S Phase ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Mitotic Kinase ◽

C Terminus ◽

N Terminus ◽

Dna Replication Checkpoint ◽

Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

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Mammalian Rif1 contributes to replication stress survival and homology-directed repair

The Journal of Cell Biology ◽

10.1083/jcb.200902039 ◽

2009 ◽

Vol 187 (3) ◽

pp. 385-398 ◽

Cited By ~ 87

Author(s):

Sara B.C. Buonomo ◽

Yipin Wu ◽

David Ferguson ◽

Titia de Lange

Keyword(s):

S Phase ◽

Replication Checkpoint ◽

Strand Breaks ◽

Cellular Processes ◽

Homology Directed Repair ◽

Conditional Deletion ◽

Dna Replication Checkpoint ◽

Mouse Cells ◽

Phase Progression ◽

Stalled Replication Forks

Rif1, originally recognized for its role at telomeres in budding yeast, has been implicated in a wide variety of cellular processes in mammals, including pluripotency of stem cells, response to double-strand breaks, and breast cancer development. As the molecular function of Rif1 is not known, we examined the consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure in embryonic development, and conditional deletion of Rif1 from mouse embryo fibroblasts affects S-phase progression, rendering cells hypersensitive to replication poisons. Rif1 deficiency does not alter the activation of the DNA replication checkpoint but rather affects the execution of repair. RNA interference to human Rif1 decreases the efficiency of homology-directed repair (HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at stalled replication forks, preferentially around pericentromeric heterochromatin. Collectively, these findings reveal a function for Rif1 in the repair of stalled forks by facilitating HDR.

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DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

The Journal of Cell Biology ◽

10.1083/jcb.200406048 ◽

2004 ◽

Vol 167 (5) ◽

pp. 841-849 ◽

Cited By ~ 15

Author(s):

Ayumi Yamada ◽

Brad Duffy ◽

Jennifer A. Perry ◽

Sally Kornbluth

Keyword(s):

Dna Replication ◽

Cell Cycle Control ◽

Checkpoint Control ◽

Checkpoint Activation ◽

Replication Checkpoint ◽

Synthetic Lethal ◽

Mitotic Entry ◽

Regulatory Module ◽

Dna Replication Checkpoint ◽

Control Modules

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7–Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

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Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e11-10-0852 ◽

2012 ◽

Vol 23 (6) ◽

pp. 1058-1067 ◽

Cited By ~ 16

Author(s):

Theresa J. Berens ◽

David P. Toczyski

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Sensor Kinase ◽

Replication Forks ◽

Checkpoint Activation ◽

Replication Checkpoint ◽

Strand Breaks ◽

Crucial Step ◽

Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

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