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

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
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.

scholarly journals The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

2009 ◽  
Vol 29 (18) ◽  
pp. 5008-5019 ◽  
Author(s):  
Makiko Komata ◽  
Masashige Bando ◽  
Hiroyuki Araki ◽  
Katsuhiko Shirahige
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Terminal Region ◽  
Methyl Methanesulfonate ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Dna Replication Checkpoint ◽  
Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

scholarly journals Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks

Oncogene ◽  
2004 ◽  
Vol 23 (21) ◽  
pp. 3872-3882 ◽  
Author(s):  
Yun-Gui Yang ◽  
Ulrich Cortes ◽  
Srinivas Patnaik ◽  
Maria Jasin ◽  
Zhao-Qi Wang
Keyword(s):  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stalled Replication Forks ◽  
Parp 1

The DNA replication checkpoint response stabilizes stalled replication forks

Nature ◽  
2001 ◽  
Vol 412 (6846) ◽  
pp. 557-561 ◽  
Author(s):  
Massimo Lopes ◽  
Cecilia Cotta-Ramusino ◽  
Achille Pellicioli ◽  
Giordano Liberi ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Dna Replication Checkpoint ◽  
Stalled Replication Forks

scholarly journals MDC1 collaborates with TopBP1 in DNA replication checkpoint control

2011 ◽  
Vol 193 (2) ◽  
pp. 267-273 ◽  
Author(s):  
Jiadong Wang ◽  
Zihua Gong ◽  
Junjie Chen
Keyword(s):  
Dna Replication ◽  
Cellular Response ◽  
Checkpoint Control ◽  
Dna Double Strand Breaks ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Major Player ◽  
Dna Replication Checkpoint ◽  
Cellular Dna ◽  
Stalled Replication Forks

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.

scholarly journals Ionizing Radiation–dependent γ-H2AX Focus Formation Requires Ataxia Telangiectasia Mutated and Ataxia Telangiectasia Mutated and Rad3-related

2005 ◽  
Vol 16 (5) ◽  
pp. 2566-2576 ◽  
Author(s):  
Joanna D. Friesner ◽  
Bo Liu ◽  
Kevin Culligan ◽  
Anne B. Britt
Keyword(s):  
Ionizing Radiation ◽  
Ataxia Telangiectasia ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Focus Formation ◽  
Strand Breaks ◽  
M Phase ◽  
Stalled Replication Forks

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (γ-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of γ-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, ∼10% of IR-induced γ-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, γ-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.

scholarly journals Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression in Escherichia coli

2007 ◽  
Vol 189 (9) ◽  
pp. 3496-3501 ◽  
Author(s):  
Estrella Guarino ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Permissive Temperature ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Progression ◽  
Holliday Junction Resolvase ◽  
Ribonucleoside Diphosphate Reductase ◽  
Stalled Replication Forks

ABSTRACT The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec+ context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named “replication fork reversal.” Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA + strain growing under limited (2-μg/ml) or under optimal (5-μg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.

scholarly journals Replication gaps underlie BRCA-deficiency and therapy response

2019 ◽  
Author(s):  
Nicholas J. Panzarino ◽  
John Krais ◽  
Min Peng ◽  
Michelle Mosqueda ◽  
Sumeet Nayak ◽  
...  
Keyword(s):  
Dna Replication ◽  
Therapy Response ◽  
Genotoxic Stress ◽  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
First Line ◽  
Strand Breaks ◽  
Genotoxic Agents ◽  
Cancer Phenotype

AbstractCancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism of action of genotoxic chemotherapies.

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

Genetics ◽  
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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