Double functions for the Mre11 complex during DNA double-strand break repair and replication

Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.     

ZIP4H (TEX11) deficiency in the mouse impairs meiotic double strand break repair and the regulation of crossing over

We have recently shown that hypomorphic Mre11 complex mouse mutants exhibit defects in the repair of meiotic double strand breaks (DSBs). This is associated with perturbation of synaptonemal complex morphogenesis, repair and regulation of crossover formation. To further assess the Mre11 complex's role in meiotic progression, we identified testis-specific NBS1-interacting proteins via two-hybrid screening in yeast. In this screen, Zip4h (Tex11), a male germ cell specific X-linked gene was isolated. Based on sequence and predicted structural similarity to the S. cerevisiae and A. thaliana Zip4 orthologs, ZIP4H appears to be the mammalian ortholog. In S. cerevisiae and A. thaliana, Zip4 is a meiosis-specific protein that regulates the level of meiotic crossovers, thus influencing homologous chromosome segregation in these organisms. As is true for hypomorphic Nbs1 (Nbs1^ΔB/ΔB) mice, Zip4h^−/Y mutant mice were fertile. Analysis of spermatocytes revealed a delay in meiotic double strand break repair and decreased crossover formation as inferred from DMC1 and MLH1 staining patterns, respectively. Achiasmate chromosomes at the first meiotic division were also observed in Zip4h^−/Y mutants, consistent with the observed reduction in MLH1 focus formation. These results indicate that meiotic functions of Zip4 family members are conserved and support the view that the Mre11 complex and ZIP4H interact functionally during the execution of the meiotic program in mammals.     

The Mre11 complex influences DNA repair, synapsis, and crossing over in murine meiosis

The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles , relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability . Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.     

The fission yeast Rad32 (Mre11)-Rad50-Nbs1 complex is required for the S-phase DNA damage checkpoint

Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G(2) DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.     

Double Strand Break Metabolism and Cancer Susceptibility: Lessons from the Mre11 Complex

Hypomorphic mutants affecting the Mre11 complex components Mre11 (Mre11ATLD1/ATLD1) and Nbs1 (Nbs1?B/?B) have been established in the mouse. These mutations recapitulate those inherited in human chromosome fragility syndromes, the ataxia-telangiectasia like disorder and Nijmegen breakage syndrome. At the cellular level, the human and murine mutants exhibit defects in the intra S and G2/M checkpoints and marked chromosome instability. Whereas these outcomes are associated with predisposition to malignancy in humans, similar predisposition was not observed in either Mre11ATLD1/ATLD1 or Nbs1?B/?B mice. These data demonstrate that chromosome breakage per se is insufficient to significantly enhance the initiation of tumorigenesis. However, these mutations greatly enhanced the risk of malignancy in p53+/- mice. We propose that proper metabolism of chromosome breaks arising during DNA replication is uniquely important for suppressing loss of heterozygosity and thus the penetrance of recessive oncogenic lesions.     

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.     

Double strand break metabolism and cancer susceptibility: lessons from the mre11 complex

Hypomorphic mutants affecting the Mre11 complex components Mre11 (Mre11(ATLD1/ATLD1)) and Nbs1 (Nbs1(DeltaB/DeltaB)) have been established in the mouse. These mutations recapitulate those inherited in human chromosome fragility syndromes, the ataxia-telangiectasia like disorder and Nijmegen breakage syndrome. At the cellular level, the human and murine mutants exhibit defects in the intra S and G2/M checkpoints and marked chromosome instability. Whereas these outcomes are associated with predisposition to malignancy in humans, similar predisposition was not observed in either Mre11(ATLD1/ATLD1) or Nbs1(DeltaB/DeltaB) mice. These data demonstrate that chromosome breakage per se is insufficient to significantly enhance the initiation of tumorigenesis. However, these mutations greatly enhanced the risk of malignancy in p53+/- mice. We propose that proper metabolism of chromosome breaks arising during DNA replication is uniquely important for suppressing loss of heterozygosity and thus the penetrance of recessive oncogenic lesions.     

DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA

The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.     

Mdm2 binds to Nbs1 at sites of DNA damage and regulates double strand break repair

Mdm2 directly regulates the p53 tumor suppressor. However, Mdm2 also has p53-independent activities, and the pathways that mediate these functions are unresolved. Here we report the identification of a specific association of Mdm2 with Mre11, Nbs1, and Rad50, a DNA double strand break repair complex. Mdm2 bound to the Mre11-Nbs1-Rad50 complex in primary cells and in cells containing inactivated p53 or p14/p19ARF, a regulator of Mdm2. Further analysis revealed that Mdm2 directly bound to Nbs1 but not to Mre11 or Rad50. Amino acids 198-314 of Mdm2 were required for Mdm2/Nbs1 association, and neither the N terminus forkhead-associated and breast cancer C-terminal domains nor the C terminus Mre11 binding domain of Nbs1 mediated the interaction of Nbs1 with Mdm2. Mdm2 co-localized with Nbs1 to sites of DNA damage following gamma-irradiation. Notably, Mdm2 overexpression inhibited DNA double strand break repair, and this was independent of p53 and ARF, the alternative reading frame of the Ink4alocus. The delay in DNA repair imposed by Mdm2 required the Nbs1 binding domain of Mdm2, but the ubiquitin ligase domain in Mdm2 was dispensable. Therefore, Nbs1 is a novel p53-independent Mdm2 binding protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.     

Dual role of Nbs1 in the ataxia telangiectasia mutated-dependent DNA damage response

The Nbs1 protein associates with Mre11 and Rad50 proteins to form the Mre11-Rad50-Nbs1 complex, which plays an important role in the intracellular signaling pathway activated in response to DNA damage. Mutations in the genes for each of these three components of the Mre11-Rad50-Nbs1 complex result in human diseases characterized by genomic instability. Insight into the functions of Nbs1 in the DNA damage response mediated by the protein kinase, ataxia telangiectasia mutated, has been provided by recent studies. Nbs1 acts both as a downstream target of ataxia telangiectasia mutated in the S-phase checkpoint of the cell cycle as well as an upstream modulator or activator of ataxia telangiectasia mutated in the DNA damage response.     

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.     

Mdm2 promotes genetic instability and transformation independent of p53

Mdm2, a regulator of the tumor suppressor p53, is frequently overexpressed in human malignancies. Mdm2 also has unresolved, p53-independent functions that contribute to tumorigenesis. Here, we show that increased Mdm2 expression induced chromosome/chromatid breaks and delayed DNA double-strand break repair in cells lacking p53 but not in cells with a mutant form of Nbs1, a component of the Mre11/Rad50/Nbs1 DNA repair complex. A 31-amino-acid region of Mdm2 was necessary for binding to Nbs1. Mutation of conserved amino acids in the Nbs1 binding domain of Mdm2 inhibited Mdm2-Nbs1 association and prevented Mdm2 from delaying phosphorylation of H2AX and ATM-S/TQ sites, repair of DNA breaks, and resolution of DNA damage foci. Similarly, the mutation of eight amino acids in the Mdm2 binding domain of Nbs1 inhibited Mdm2-Nbs1 interaction and blocked the ability of Mdm2 to delay DNA break repair. Both Nbs1 and ATM, but not the ubiquitin ligase activity of Mdm2, were necessary to inhibit DNA break repair. Only Mdm2 with an intact Nbs1 binding domain was able to increase the frequency of chromosome/chromatid breaks and the transformation efficiency of cells lacking p53. Therefore, the interaction of Mdm2 with Nbs1 inhibited DNA break repair, leading to chromosome instability and subsequent transformation that was independent of p53.     

Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells

Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ.     

Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11

Mre11-Rad50-Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.     

Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance

The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.     

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.     

Linkage between Werner syndrome protein and the Mre11 complex via Nbs1

The Werner syndrome and the Nijmegen breakage syndrome are recessive genetic disorders that show increased genomic instability, cancer predisposition, hypersensitivity to mitomycin C and gamma-irradiation, shortened telomeres, and cell cycle defects. The protein mutated in the premature aging disease known as the Werner syndrome is designated WRN and is a member of the RecQ helicase family. The Nbs1 protein is mutated in Nijmegen breakage syndrome individuals and is part of the mammalian Mre11 complex together with the Mre11 and Rad50 proteins. Here, we show that WRN associates with the Mre11 complex via binding to Nbs1 in vitro and in vivo. In response to gamma-irradiation or mitomycin C, WRN leaves the nucleoli and co-localizes with the Mre11 complex in the nucleoplasm. We detect an increased association between WRN and the Mre11 complex after cellular exposure to gamma-irradiation. Small interfering RNA and complementation experiments demonstrated convergence of WRN and Nbs1 in response to gamma-irradiation or mitomycin C. Nbs1 is required for the Mre11 complex promotion of WRN helicase activity. Taken together, these results demonstrate a functional link between the two genetic diseases with partially overlapping phenotypes in a pathway that responds to DNA double strand breaks and interstrand cross-links.     

Structural and functional analysis of Mre11-3

The Mre11, Rad50 and Nbs1 proteins make up the conserved multi-functional Mre11 (MRN) complex involved in multiple, critical DNA metabolic processes including double-strand break repair and telomere maintenance. The Mre11 protein is a nuclease with broad substrate recognition, but MRN-dependent processes requiring the nuclease activity are not clearly defined. Here, we report the functional and structural characterization of a nuclease-deficient Mre11 protein termed mre11-3. Importantly, the hmre11-3 protein has wild-type ability to bind DNA, Rad50 and Nbs1; however, nuclease activity was completely abrogated. When expressed in cell lines from patients with ataxia telangiectasia-like disorder (ATLD), hmre11-3 restored the formation of ionizing radiation-induced foci. Consistent with the biochemical results, the 2.3 Å crystal structure of mre11-3 from Pyrococcus furiosus revealed an active site structure with a wild-type-like metal-binding environment. The structural analysis of the H85L mutation provides a detailed molecular basis for the ability of mre11-3 to bind but not hydrolyze DNA. Together, these results establish that the mre11-3 protein provides an excellent system for dissecting nuclease-dependent and independent functions of the Mre11 complex.     

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.