Abstract: Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNA-end binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.

Abstract: Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.

Abstract: The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

Abstract: Purpose To investigate the expression and predictive role of the Mre11/Rad50/Nbs1 (MRN) complex and the ataxia-telangiectasia mutated protein (ATM) for the outcome of radiotherapy in breast cancer patients. Methods and Materials The protein expression of ATM and the DNA repair proteins in the MRN complex were investigated using immunohistochemistry in tumors from 224 women with early breast cancer, who were randomized to receive postoperative radiotherapy or adjuvant chemotherapy. Results Compared with normal breast tissue, the staining intensity of Mre11, Rad50, Nbs1, and ATM was reduced in a majority of the tumors. Weak expression of the MRN complex was correlated with high histologic grade and estrogen receptor negativity (p = 0.01 and p = 0.0001, respectively). Radiotherapy significantly reduced the risk of local recurrence as compared with chemotherapy (p = 0.04). The greatest benefit of radiotherapy was seen in patients with moderate/strong expression of the MRN complex (relative risk = 0.27, 95% confidence interval = 0.098–0.72, p = 0.009), whereas patients with negative/weak MRN expression had no benefit of radiotherapy compared with adjuvant chemotherapy. These results suggest that an intact MRN complex is important for the tumor cell eradicating effect of radiotherapy. Conclusions Reduced expression of the MRN complex predicts a poor effect of radiotherapy in patients with early breast cancer.

Abstract: In response to DNA damage, cells activate a complex protein network designed to sustain genomic integrity. Many of the proteins involved in the network form discrete repair foci, the composition of which is determined by the specific type of damage. Replication protein A (RPA) and the Mre11/Rad50/Nbs1 (MRN) complex both participate in foci and co-localize at certain types of lesions. Following etoposide (ETOP) treatment, cells form foci containing either RPA or the MRN complex, but not both. To investigate this preferential foci formation, we used cell cycle synchronization experimentation. We demonstrate that cells in S phase contain RPA foci but lack phospho-Nbs1 foci. This is consistent with RPA's role in homologous recombination repair of DNA double-strand breaks (DSBs), the predominant form of repair during S phase. Cells synchronized at G0/G1 phase contain phospho-Nbs1 foci, consistent with the MRN complex involvement in non-homologous end joining, the predominant form of repair in G1 phase. Treatment of cells with the proteasome inhibitor MG132 dramatically reduced the percentage of cells forming phospho-Nbs1 foci but did not alter the percentage of cells containing RPA or phospho-RPA foci. ETOP induced similar amounts of damage in all phases of the cell cycle as measured by the comet assay. These data suggest that in response to DNA DSBs, cell cycle-preferred repair pathways differentially engage RPA and the MRN complex in repair foci.

Abstract: The presence of DNA damage activates a specific response cascade culminating in DNA repair activity and cell cycle checkpoints. Although the type of lesion dictates what proteins are involved in the response, replication protein A (RPA) and the Mre11/ Rad50/Nbs1 complex (MRN) respond to most types of lesions. To examine the relationship of RPA and the MRN complex in DNA damage responses, we used siRNA-mediated protein depletion of RPA-p70 and Mre11. Depletion of RPA-p70 decreased the ability of cells to form phospho-Nbs1 foci and increased levels of DNA double-strand breaks (DSBs) following treatment with etoposide (ETOP). In contrast, depletion of Mre11 led to increased levels of RPA-p34 foci formation, but abrogated phospho-RPA-p34 foci formation. These data support a role for RPA as an initial signal/sensor for DNA damage that facilitates recruitment of MRN and ATM/ATR to sites of damage, where they then work together to fully activate the DNA damage response.

Abstract: Adeno-associated virus type 2 (AAV-2) is a nonpathogenic, replication defective parvovirus containing a single-stranded DNA genome of 4.7 kb. Despite the increasing utilization of recombinant vectors derived from this virus (rAAV) in gene transfer applications, several aspects of the biology of both the wild type virus and of its vectors remain poorly understood. Here we developed a technique to visualize the conversion of rAAV vector genomes from single-stranded (ss) to double-stranded (ds) DNA in real time. We report that rAAV DNA accumulates into discrete foci inside the nucleus. These rAAV foci are defined in number, increase in size over time after transduction, and their presence correlates with the efficiency of cell transduction. These structures overlap with, or lie in close proximity to, the foci in which proteins of the MRN (Mrell-Rad50-Nbsl) complex and Mdcl accumulate after DNA damage. Silencing of Nbsl or Mdcl by RNA interference markedly increases the formation of rAAV foci, the extent of rAAV transduction, and AAV Rep dependent site-specific integration at chromosome 19q.l3.3-qter, so called AAVS1 locus. The adenovirus E4orf6 and ElB55k proteins-mediated degradation of the MRN complex also correlated with high levels of rAAV transduction and foci formation. Taken together, these observations indicate that the MRN complex plays an inhibitory role at the level of rAAV ss to double-stranded DNA (dsDNA) genome conversion, vector transduction, and site-specific integration. On the other hand, similar experiments using siRNAs against histone H2AX, Rad52, and DNA-PKcs indicated that these factors are required for effective rAAV transduction and site-specific integration.

Abstract: The proapoptotic protein Bid is phosphorylated by ATM after double strand breaks (DSBs) induction and induces S-phase arrest by a mechanism that remains to be elucidated. Here we show that in mammalian cells, Bid is associated with Mre11, a subunit of the Mre11-Rad50-Nbs1 (MRN) complex. We demonstrate that Bid activation is abrogated in Mre11 and Nbs1 deficient primary mouse fibroblasts and cells from patients with ataxia talangiectasia-like disorder. Bid depletion by RNA interference inhibited the S-phase checkpoint activation and G2/M arrest after genotoxic insult, but had no effect on MRN complex formation. Our results explain the mechanism of Bid phosphorylation by ATM in response to DNA damage and suggest that Bid functions as a link between the MRN complex and S-phase regulatory proteins.

Abstract: The MRN complex constitutes of MRE11,NBS1 and RAD50,and the deficiency of MRE11 and NBS1 caused disorder named ataxia-telangiectasia-like disease(ATLD) and Nijmegen breakage syndrome(NBS),respectively.MRN complex is a central player in most aspects of the cellular response to DNA double-strand breaks,and also function in homologous recombination,non-homologous recombination,telomere maintenance,DNA damage checkpoint activation and DNA replication insurance.Here,recent finding about MRN complex was reviewed.

Abstract: The Mre11–Rad50–Nbs1 (MRN) complex is providing paradigm-shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DNA double-strand breaks (DSBs), and MRN mutations cause the human cancer predisposition diseases Nijmegen breakage syndrome and ataxia telangiectasia-like disorder (ATLD). MRN’s 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome (SMC) related protein, employs its ATP-binding cassette (ABC) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. Contributing to MRN regulatory roles, Nbs1 harbors N-terminal phosphopeptide interacting FHA and BRCT domains, as well as C-terminal ataxia telangiectasia mutated (ATM) kinase and Mre11 interaction domains. Current emerging structural and biological evidenc...

Abstract: Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.

Abstract: The ability of plants to repair DNA double-strand breaks (DSBs) is essential for growth and fertility. The Arabidopsis DSB repair proteins AtRAD50 and AtMRE11 form part of an evolutionarily conserved complex that, in Saccharomyces cerevisiae and mammals, includes a third component termed XRS2 and NBS1, respectively. The MRN complex (MRX in yeast) has a direct role in DSB repair and is also required for DNA damage signaling and checkpoint activation in a pathway mediated by the protein kinase ATM. This study characterizes Arabidopsis and maize NBS1 orthologues that share conserved protein motifs with human NBS1. Both plant NBS1 proteins interact with the corresponding MRE11 orthologues, and deletion analysis of AtNBS1 defines a region towards the C-terminus (amino acids 465-500) that is required for interaction with AtMRE11. Arabidopsis lines homozygous for a T-DNA insertional mutation in AtNBS1 display hypersensitivity to the DNA cross-linking reagent mitomycin C, and this phenotype can be rescued by complementation with the wild-type gene, consistent with a function for AtNBS1 in plant DSB repair. Analysis of atnbs1-1 atatm double mutants revealed a role for AtNBS1 in meiotic recombination. While atatm mutants produce reduced seed numbers, plants deficient in both AtATM and AtNBS1 are completely infertile. Cytological analysis of these double mutants revealed incomplete chromosome pairing and synapsis in meiotic prophase, and extensive chromosome fragmentation in metaphase I and subsequent stages. These results suggest a novel role for AtNBS1 that is independent of AtATM-mediated signaling and functions in the very early stages of meiosis.

Abstract: The recognition and repair of DNA double-strand breaks (DSBs) is a complex process that draws upon a multitude of proteins. This is not surprising since this is a lethal lesion if left unrepaired and also contributes to genome instability and the consequential risk of cancer and other pathologies. Some of the key proteins that recognize these breaks in DNA are mutated in distinct genetic disorders that predispose to agent sensitivity, genome instability, cancer predisposition and/or neurodegeneration. These include members of the Mre11 complex (Mre11/Rad50/Nbs1) and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human genetic disorder A-T. The mre11 (MRN) complex appears to be the major sensor of the breaks and subsequently recruits ATM where it is activated to phosphorylate in turn members of that complex and a variety of other proteins involved in cell-cycle control and DNA repair. The MRN complex is also upstream of ATM and ATR (A-T-mutated and rad3-related) protein in responding to agents that block DNA replication. To date, more than 30 ATM-dependent substrates have been identified in multiple pathways that maintain genome stability and reduce the risk of disease. We focus here on the relationship between ATM and the MRN complex in recognizing and responding to DNA DSBs.