Choreography of the DNA Damage Response

Lisby, Michael; Barlow, Jacqueline H.; Burgess, Rebecca C.; Rothstein, Rodney

doi:10.1016/j.cell.2004.08.015

Cited by 824 publications

(464 citation statements)

References 85 publications

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“…In addition, many proteins are known to localize to the site of DNA damage in cells (43). Thus, an understanding of the protein-protein interactions for DNA damage and replication checkpoint adaptor proteins is needed to understand the specificity of DNA damage and replication checkpoint signaling in vivo.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Chen¹,

Zhou

2009

Journal of Biological Chemistry

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Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1-3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4 -7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR, 2 is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single-and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were foun...

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Section: Discussionmentioning

confidence: 99%

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Chen¹,

Zhou

2009

Journal of Biological Chemistry

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“…In yeast Saccharomyces cerevisiae, it involves (i) initial DSB processing by MRX(Mre11‐Rad50‐Xrs2)/Sae2 producing a short 3′ overhang; (ii) long‐range DNA resection by two redundant machineries, Dna2/Sgs1‐Top3‐Rmi1 and Exo1 nuclease (Mimitou & Symington, 2008; Zhu et al , 2008), which generate long tracts of ssDNA covered by the ssDNA‐binding protein RPA and required for DNA damage checkpoint activation and loading of homologous recombination machinery (Zou & Elledge, 2003; Lisby et al , 2004); (iii) loading of the homologous recombination protein Rad52 followed by recruitment of Rad51 which generates a nucleoprotein filament stabilized by Rad55/57 (Symington et al , 2014). During HR and BIR, Rad52/51/55/57 promote homology search and invasion of intact donor dsDNA by the processed broken end to initiate repair (Anand et al , 2013; Symington et al , 2014).…”

Section: Introductionmentioning

confidence: 99%

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.

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“…Foci are easily observed because many DNA repair proteins are retained at DSBs in suprastoichiometric ratios. It has been estimated that individual breaks contains at least 1,000 molecules of each DNA repair protein (Lisby et al, 2004), revealing that many repair proteins are concentrated at repair sites by a factor of 10 3 . The reasons for this stoichiometry are unknown, although it is speculated that high local concentrations of repair or linkages between ubiquitin moieties can lead to vastly different biological outcomes.…”

Section: Principles Of Dsb Recognitionmentioning

confidence: 99%