The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.     

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.     

The Mre11/Rad50/Nbs1 complex plays an important role in the prevention of DNA rereplication in mammalian cells

The Mre11/Nbs1/Rad50 complex (MRN) plays multiple roles in the maintenance of genome stability, including repair of double-stranded breaks (DSBs) and activation of the S-phase checkpoint. Here we demonstrate that MRN is required for the prevention of DNA rereplication in mammalian cells. DNA replication is strictly regulated by licensing control so that the genome is replicated once and only once per cell cycle. Inactivation of Nbs1 or Mre11 leads to a substantial increase of DNA rereplication induced by overexpression of the licensing factor Cdt1. Our studies reveal that multiple mechanisms are likely involved in the MRN-mediated suppression of rereplication. First, both Mre11 and Nbs1 are required for facilitating ATR activation when Cdt1 is overexpressed, which in turn suppresses rereplication. Second, Cdt1 overexpression induces ATR-mediated phosphorylation of Nbs1 at Ser343 and this phosphorylation depends on the FHA and BRCT domains of Nbs1. Mutations at Ser343 or in the FHA and BRCT domains lead to more severe rereplication when Cdt1 is overexpressed. Third, the interaction of the Mre11 complex with RPA is important for the suppression of rereplication. This suggests that modulating RPA activity via a direct interaction of MRN is likely one of the effector mechanisms to suppress rereplication. Moreover, we demonstrate that MRN is also required for preventing the accumulation of DSBs when rereplication is induced. Therefore, our studies suggest new roles of MRN in the maintenance of genome stability through preventing rereplication and rereplication-associated DSBs when licensing control is compromised.     

A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.     

Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock

Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.     

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.     

The Mre11/Rad50/Nbs1 Complex and Its Role as a DNA Double-Strand Break Sensor for ATM

Abstract not yet available.     

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.     

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.     

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.     

DNA lesion-specific co-localization of the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA) to repair foci

The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci were detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions.     

Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.     

Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes

Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.     

The Rad50 hook domain is a critical determinant of Mre11 complex functions

The Mre11 complex (in Saccharomyces cerevisiae: Mre11, Rad50 and Xrs2) influences multiple facets of chromosome break metabolism. A conserved feature of the Mre11 complex is a zinc-coordinating motif in Rad50 called the Rad50 hook. We established a diploid yeast strain, rad50(hook), in which Rad50 is encoded in halves, one from each of the two RAD50 alleles, with the residues constituting the hook deleted. In all respects, rad50(hook) phenocopies complete Rad50 deficiency. Replacing the hook domain with a ligand-inducible FKBP dimerization cassette partially mitigated all phenotypes in a ligand-dependent manner. The data indicate that the Rad50 hook is critical for Mre11 complex-dependent DNA repair, telomere maintenance and meiotic double-strand break formation. Sister chromatid cohesion was unaffected by Rad50 deficiency, suggesting that molecular bridging required for recombinational DNA repair is qualitatively distinct from cohesin-mediated sister chromatid cohesion.     

DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP

The Rad50, Mre11 and Nbs1 complex is involved in many essential chromosomal organization processes dealing with DNA ends, including two major pathways of DNA double-strand break repair, homologous recombination and non-homologous end joining. Previous data on the structure of the human Rad50 and Mre11 (R/M) complex suggest that a common role for the protein complex in these processes is to provide a physical link between DNA ends such that they can be processed in an organized and coordinated manner. Here we describe the DNA binding properties of the R/M complex. The complex bound to both single-stranded and double-stranded DNA. Scanning force microscopy analysis of DNA binding by R/M showed the requirement for an end to form oligomeric R/M complexes, which could then migrate or transfer away from the end. The R/M complex had a lower preference for DNA substrates with 3′-overhangs compared with blunt ends or 5′-overhangs. Interestingly, ATP binding, but not hydrolysis, increased the preference of R/M binding to DNA substrates with 3′-overhangs relative to substrates with blunt ends and 5′-overhangs.     

Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes

The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans. In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses. Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities. Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes. Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release. The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association. The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays. Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.     

Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex

Studies of human Nijmegen breakage syndrome (NBS) cells have led to the proposal that the Mre11/Rad50/ NBS1 complex, which is involved in the repair of DNA double-strand breaks (DSBs), might also function in activating the DNA damage checkpoint pathways after DSBs occur. We have studied the role of the homologous budding yeast complex, Mre11/Rad50/Xrs2, in checkpoint activation in response to DSB-inducing agents. Here we show that this complex is required for phosphorylation and activation of the Rad53 and Chk1 checkpoint kinases specifically in response to DSBs. Consistent with defective Rad53 activation, we observed defective cell-cycle delays after induction of DSBs in the absence of Mre11. Furthermore, after gamma-irradiation phosphorylation of Rad9, which is an early event in checkpoint activation, is also dependent on Mre11. All three components of the Mre11/Rad50/Xrs2 complex are required for activation of Rad53, however, the Ku80, Rad51 or Rad52 proteins, which are also involved in DSB repair, are not. Thus, the integrity of the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after the formation of DSBs.     

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.