ATP‐dependent DNA binding,unwinding, and resection by the Mre11/Rad50 complex

ATP‐dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double‐strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MR‐ATPγS‐DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS‐bound Rad50 nucleotide‐binding domains. Duplex DNA cannot access the Mre11 active site in the ATP‐free full‐length MR complex. ATP hydrolysis drives rotation of the nucleotide‐binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis‐driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.     

Structure of the Rad50 DNA double‐strand break repair protein in complex with DNA

The Mre11–Rad50 nuclease–ATPase is an evolutionarily conserved multifunctional DNA double‐strand break (DSB) repair factor. Mre11–Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50^NBD (nucleotide‐binding domain) in complex with Mre11^HLH (helix‐loop‐helix domain), AMPPNP, and double‐stranded DNA. DNA binds between both coiled‐coil domains of the Rad50 dimer with main interactions to a strand‐loop‐helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double‐strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP‐bound state.     

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50

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Taking the time to make important decisions: The checkpoint effector kinases Chk1 and Chk2 and the DNA damage response

The cellular DNA damage response (DDR) is activated by many types of DNA lesions. Upon recognition of DNA damage by sensor proteins, an intricate signal transduction network is activated to coordinate diverse cellular outcomes that promote genome integrity. Key components of the DDR in mammalian cells are the checkpoint effector kinases Chk1 and Chk2 (referred to henceforth as the effector kinases; orthologous to spChk1 and spCds1 in the fission yeast S. pombe and scChk1 and scRad53 in the budding yeast S. cerevisiae). These evolutionarily conserved and structurally divergent kinases phosphorylate numerous substrates to regulate the DDR. This review will focus on recent advances in our understanding of the structure, regulation, and functions of the effector kinases in the DDR, as well as their potential roles in human disease.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.     

Prognostic implication and functional annotations of Rad50 expression in patients with prostate cancer

Increasing evidence has shown that Rad50, a protein involved in the DNA damage repair process, significantly correlated with tumor prognosis. This study focused on Rad50 expression in tumor samples and its prognostic value for patients with prostate cancer (PCa). In this study, significantly elevated Rad50 expression in PCa tissues compared to normal tissues (P < .01). Five independent Oncomine databases validated significant differential expression of Rad50 (P < .001). Hence, 80 patients with PCa from Fudan University Shanghai Cancer Center (FUSCC) and 351 patients with PCa with available protein expression data from The Cancer Genome Atlas (TCGA) were included to investigate the survival benefit. Univariate and multivariate Cox regression analyses were performed to investigate the significance of clinicopathological factors on disease-free survival (DFS) and overall survival (OS). Kaplan-Meier analysis indicated that elevated Rad50 protein expression levels significantly correlated with unfavorable DFS (P = .005) in the FUSCC cohort and poorer OS (P = .04) in TCGA cohort. Furthermore, coregulation analysis of proteins indicated that 76 coregulated proteins were associated with Rad50, while 11 most highly involved hub proteins, including Rad50, MRE11A, DUT, POLR3A, MCM3AP, RECQL, PNPT1, RANBP3, DDX1, SNRPB, and UGN, were significantly coregulated in the protein-protein interaction network. Functional enrichment analysis consecutively indicated significant functions and signaling pathways including DNA replication, spliceosome, DNA geometric change, homologous recombination, and G2M checkpoint. This study first reveals that elevated Rad50 expression is significantly associated with aggressive progression and poor survival for patients with PCa. Together, these data suggest that Rad50 may act as an oncoprotein, guide the molecular diagnosis, and may shed light on novel individual therapeutic strategies for progressive PCa patients.     

A network of allosterically coupled residues in the bacteriophage T4 Mre11–Rad50 complex

The Mre11–Rad50 (MR) protein complex, made up of a nuclease and ATPase, respectively, is involved in the processing of double‐strand breaks as part of an intricate mechanism for their repair. Although it is clear that the MR complex is subject to allosteric regulation and that there is communication between the nuclease and ATPase active sites, the underlying mechanisms are poorly understood. We performed statistical coupling analysis on Mre11 and Rad50 to predict linked residues based on their evolutionary correlation. This analysis predicted a coevolving sector of six residues that may be allosterically coupled. The prediction was tested using double‐mutant cycle analysis of nuclease and ATPase activity. The results indicate that a tyrosine residue located near the active site of Mre11 is allosterically coupled to several Rad50 residues located over 40 Å away. This allosteric coupling may be the basis for the reciprocal regulation of the ATPase and nuclease activities of the complex.     

Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.     

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.     

Rad50S alleles of the Mre11 complex: questions answered and questions raised

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; D'D. Amours, S.P. Jackson, The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15 (2001) 2238-49. ; M. Grenon, C. Gilbert, N.F. Lowndes, Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3 (2001) 844-847. ] where the signaling is compromised. Herein, we describe evidence for chronic signaling by Rad50S and discuss possible mechanisms.     

Cdk1-dependent regulation of the Mre11 complex couples DNA repair pathways to cell cycle progression

Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways ensuring the repair of DNA double-stranded breaks (DSBs) in eukaryotes. It has long been known that cell cycle stage is a major determinant of the type of pathway used to repair DSBs in vivo. However, the mechanistic basis for the cell cycle regulation of the DNA damage response is still unclear. Here we show that a major DSB sensor, the Mre11–Rad50–Xrs2 (MRX) complex, is regulated by cell cycle-dependent phosphorylation specifically in mitosis. This modification depends on the cyclin-dependent kinase Cdc28/Cdk1, and abrogation of Xrs2 and Mre11 phosphorylation results in a marked preference for DSB repair through NHEJ. Importantly, we show that phosphorylation of the MRX complex after DNA damage and during mitosis are regulated independently of each other by Tel1/ATM and Cdc28/Cdk1 kinases. Collectively, our results unravel an intricate network of phosphoregulatory mechanisms that act through the MRX complex to modulate DSB repair efficiency during mitosis.     

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.     

The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11(RK) protein devoid of methylated arginines. The Mre11(RK/RK) mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11(RK/RK) MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M(RK)RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M(RK)RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.