Choreography of the DNA damage response: Spatiotemporal relationships among checkpoint and repair proteins
TL;DR: The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.
About: This article is published in Cell.The article was published on 2004-09-17 and is currently open access. It has received 938 citations till now. The article focuses on the topics: DNA repair & G2-M DNA damage checkpoint.
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TL;DR: HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified.
Abstract: Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.
1,542 citations
TL;DR: The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.
Abstract: The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.
1,389 citations
TL;DR: The components of the end resection machinery, the role of end structure, and the cell-cycle phase on resection and the interplay of end processing with NHEJ are reviewed.
Abstract: DNA double-strand breaks (DSBs) are cytotoxic lesions that can result in mutagenic events or cell death if left unrepaired or repaired inappropriately. Cells use two major pathways for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). The choice between these pathways depends on the phase of the cell cycle and the nature of the DSB ends. A critical determinant of repair pathway choice is the initiation of 5′-3′ resection of DNA ends, which commits cells to homology-dependent repair, and prevents repair by classical NHEJ. Here, we review the components of the end resection machinery, the role of end structure, and the cell-cycle phase on resection and the interplay of end processing with NHEJ.
1,363 citations
Cites background from "Choreography of the DNA damage resp..."
...Mre11 and Rad50 are conserved in prokaryotes, archeae, and eukaryotes, whereas Xrs2/Nbs1 is found only in eukaryotes and functions to signal DSBs via the PI3K-like kinase (PIKK), Tel1/ATM (ataxia telangiectasia mutated) (136, 147)....
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...Xrs2/Nbs1 is the least conserved member of the MRX/N complex and is thought to act as a regulatory and protein recruitment module (147)....
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...Upon Sae2/Ctp1 depletion, the MRX/N complex is stabilized at DSBs, which could promote sustained Tel1 signaling via interaction with Xrs2/Nbs1 (37, 87, 88)....
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...Both Rad50 and Xrs2/Nbs1 enhance the nuclease activity of Mre11 in vitro (121, 122, 152)....
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...However, localization of RPA to discrete subnuclear sites (foci) can be used as an indirect measure of resection (9, 88)....
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TL;DR: These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
Abstract: In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
1,306 citations
TL;DR: The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity and has provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases.
Abstract: The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.
1,132 citations
References
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TL;DR: The analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions, which contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
Abstract: Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
4,895 citations
"Choreography of the DNA damage resp..." refers background in this paper
...Taken together, the simultaneous presence of multiple checkpoint and (Figure 2D and Supplemental Figures S2A–S2B available on Cell website) (Gavin et al., 2002; Hays et al., 1998;recombination proteins at damaged sites suggests that DSB repair is a concerted process rather than a one Kim and Brill,…...
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TL;DR: The data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.
Abstract: The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.
2,632 citations
"Choreography of the DNA damage resp..." refers background or methods in this paper
...…observed for the recruit-To test the interdependencies of repair and checkpoint ment of ATR/ATRIP in Xenopus extracts and in mamma-proteins during their recruitment to sites of DNA damlian cells (Costanzo et al., 2003; Zou and Elledge, 2003a).age, we performed a genetic analysis of focus formation....
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...This notion has previously been suggested by an inability of a checkpoint defective rfa1-by one association/dissociation of each protein sub- complex. t11 mutant to recruit Ddc1 and Ddc2 to single-stranded DNA as measured by chromatin immunoprecipitation (Zou and Elledge, 2003a; Zou et al., 2003b)....
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TL;DR: It is suggested that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination.
1,227 citations
"Choreography of the DNA damage resp..." refers background in this paper
...Tel1not shown) (Hays et al., 1995; Shinohara et al., 1992). foci are strictly dependent on each of the subunits ofRad55 focus formation is dependent on Rad51 and the MRX complex but independent of all other proteinsRad52 but not on Rdh54, Rad59, or Rad54 (Figure 2B). tested (Figure 2D), providing…...
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TL;DR: It is shown that functional MRN is required for ATM activation, and consequently for timely activation of ATM‐mediated pathways, and explain the clinical resemblance between A‐T and A-TLD.
Abstract: The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.
1,093 citations
TL;DR: An overview of recent biochemical and structural analyses of the Rad52 group proteins is provided and how this information can be incorporated into genetic studies of recombination is discussed.
Abstract: The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
1,012 citations
"Choreography of the DNA damage resp..." refers background or methods in this paper
...…Surgeons ber of steps and involves proteins such as replication701 West 168th Street protein A (RP-A consisting of the Rfa1-3 subunits),New York, New York 10032 Rad51, Rad52, Rad54, Rdh54(Tid1), Rad55, Rad57, and2 Department of Biological Sciences Rad59 (Game and Mortimer, 1974; Symington, 2002)....
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...As measured by BrdU incorporation, the Toczyski, 2002; Symington, 2002)....
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