Mre11 complex and DNA replication: linkage to E2F and sites of DNA synthesis.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

A DNA damage response pathway controlled by Tel1 and the Mre11 complex.

We define a DNA damage checkpoint pathway in S. cerevisiae governed by the ATM homolog Tel1 and the Mre11 complex. In mitotic cells, the Tel1-Mre11 complex pathway triggers Rad53 activation and its interaction with Rad9, whereas in meiosis it acts via Rad9 and the Rad53 paralog Mre4/Mek1. Activation of the Tel1-Mre11 complex pathway checkpoint functions appears to depend upon the Mre11 complex as a damage sensor and, at least in meiotic cells, to depend on unprocessed DNA double-strand breaks (DSBs). The DSB repair functions of the Mre11 complex are enhanced by the pathway, suggesting that the complex both initiates and is regulated by the Tel1-dependent DSB signal. These findings demonstrate that the diverse functions of the Mre11 complex in the cellular DNA damage response are conserved in mammals and yeast.

An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele.

Nijmegen breakage syndrome (NBS) is a rare chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity and radioresistant DNA synthesis-S phase checkpoint deficiency, which results in the failure to suppress DNA replication origins following DNA damage. Approximately 90% of NBS patients are homozygous for the 657del5 allele, a truncating mutation of NBS1 that causes premature termination at codon 219. Because null mutations in MRE11 and RAD50, which encode binding partners of NBS1, are lethal in vertebrates, and mouse Nbs1-null mutants are inviable, we tested the hypothesis that the NBS1 657del5 mutation was a hypomorphic defect. We showed that NBS cells contain the predicted 26-kD amino-terminal protein fragment, NBS1p26, and a 70-kD NBS1 protein (NBS1p70) lacking the native N terminus. The NBSp26 protein is not physically associated with the MRE11 complex, whereas the p70 species is physically associated with it. NBS1p70 is produced by internal translation initiation within the NBS1 mRNA using an open reading frame generated by the 657del5 frameshift. We propose that the common NBS1 allele encodes a partially functional protein that diminishes the severity of the NBS phenotype.

DNA damage-dependent nuclear dynamics of the Mre11 complex.

The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.

Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.

The Mre11 complex and ATM: collaborating to navigate S phase.

Recently, findings regarding a group of cancer predisposition and chromosome instability syndromes, Nijmegen breakage syndrome (NBS), the ataxia-telangiectasia-like disorder (A-TLD) and ataxia telangiectasia have shed light on the unexpected role of recombinational DNA repair proteins in DNA-damage-dependent cell-cycle regulation. Mutations in the Mre11 complex cause A-TLD and NBS. In addition, functions of the Mre11 complex have been biochemically linked to ATM, the large protein kinase that is defective in ataxia-telangiectasia cells by the observation that Nbs1 is a bona fide substrate of the ATM kinase.

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder.

We show that hypomorphic mutations in hMRE11, but not in ATM, are present in certain individuals with an ataxia-telangiectasia-like disorder (ATLD). The cellular features resulting from these hMRE11 mutations are similar to those seen in A-T as well as NBS and include hypersensitivity to ionizing radiation, radioresistant DNA synthesis, and abrogation of ATM-dependent events, such as the activation of Jun kinase following exposure to gamma irradiation. Although the mutant hMre11 proteins retain some ability to interact with hRad50 and Nbs1, formation of ionizing radiation-induced hMre11 and Nbs1 foci was absent in hMRE11 mutant cells. These data demonstrate that ATM and the hMre11/hRad50/Nbs1 protein complex act in the same DNA damage response pathway and link hMre11 to the complex pathology of A-T.

The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.

Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.

The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair. Whereas genetic analyses in Saccharomyces cerevisiae have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions. We established mRad50 mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response. Early embryonic cells deficient in mRad50 are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs. However, the null mrad50 mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability.

Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.

Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair.

The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response.

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions.

In situ visualization of DNA double-strand break repair in human fibroblasts.

A method was developed to examine DNA repair within the intact cell. Ultrasoft x-rays were used to induce DNA double-strand breaks (DSBs) in defined subnuclear volumes of human fibroblasts and DNA repair was visualized at those sites. The DSBs remained in a fixed position during the initial stages of DNA repair, and the DSB repair protein hMre11 migrated to the sites of damage within 30 minutes. In contrast, hRad51, a human RecA homolog, did not localize at sites of DNA damage, a finding consistent with the distinct roles of these proteins in DNA repair.

hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.

We previously identified a conserved multiprotein complex that includes hMre11 and hRad50. In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair. hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation. hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51. Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both. The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells. hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased. These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair. Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.

The RAD52 epistasis group in mammalian double strand break repair.

The S. cerevisiae RAD52 epistasis group gene products mediate DNA double strand break repair and recombination. These proteins and their modes of action have been extensively characterized. The existence of highly conserved mammalian RAD52 epistasis group homologues suggests that information regarding the functions and mechanisms of double strand break repair proteins in yeast may be applicable to mammalian recombinational DNA repair. Herein, we provide an overview of the S. cerevisiae RAD52 epistasis group and describe the characterization of the five mammalian RAD52 epistasis group homologues identified to date. In the context of their expression patterns and other functional analyses, we discuss potential roles for these proteins in mammalian recombinational DNA repair and specialized recombination events such as V(D)J recombination.