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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SMC (structural maintenance of chromosomes) proteins are large coiled-coil proteins involved in chromosome condensation, sister chromatid cohesion, and DNA double-strand break processing. They share a conserved five-domain architecture with three globular domains separated by two long coiled-coil segments. The coiled-coil segments are antiparallel, bringing the N and C-terminal globular domains together. We have expressed a fusion protein of the N and C-terminal globular domains of Thermotoga maritima SMC in Escherichia coli by replacing the approximately 900 residue coiled-coil and hinge segment with a short peptide linker. The SMC head domain (SMChd) binds and condenses DNA in an ATP-dependent manner. Using selenomethionine-substituted protein and multiple anomalous dispersion phasing, we have solved the crystal structure of the SMChd to 3.1 A resolution. In the monoclinic crystal form, six SMChd molecules form two turns of a helix. The fold of SMChd is closely related to the ATP-binding cassette (ABC) ATPase family of proteins and Rad50, a member of the SMC family involved in DNA double-strand break repair. In SMChd, the ABC ATPase fold is formed by the N and C-terminal domains with the 900 residue coiled-coil and hinge segment inserted in the middle of the fold. The crystal structure of an SMChd confirms that the coiled-coil segments in SMC proteins are anti-parallel and shows how the N and C-terminal domains come together to form an ABC ATPase. Comparison to the structure of the MukB N-terminal domain demonstrates the close relationship between MukB and SMC proteins, and indicates a helix to strand conversion when N and C-terminal parts come together.
J Mol Biol 2001 Feb 09
PMID:Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted. 1117 91

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.
Mol Cell Biol 2001 Mar
PMID:Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization. 1123 51

The majority of antigen receptor diversity in mammals is generated by V(D)J recombination. During this process DNA double strand breaks are introduced at recombination signals by lymphoid specific RAG1/2 proteins generating blunt ended signal ends and hairpinned coding ends. Rejoining of all DNA ends requires ubiquitously expressed DNA repair proteins, such as Ku70/86 and DNA ligase IV/XRCC4. In addition, the formation of coding joints depends on the function of the scid gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK(CS), that is somehow required for processing of coding end hairpins. Recently, it was shown that purified RAG1/2 proteins can cleave DNA hairpins in vitro, but the same activity was also described for a protein complex of the DNA repair proteins Nbs1/Mre11/Rad50. This leaves the possibility that either protein complex might be involved in coding end processing in V(D)J recombination. We have therefore analyzed V(D)J recombination in cells from patients with Nijmegen breakage syndrome, carrying a mutation in the nbs1 gene. We find that V(D)J recombination frequencies and the quality of signal and coding joining are comparable to wild-type controls, as analyzed by a cellular V(D)J recombination assay. In addition, we did not detect significant differences in CDR3 sequences of endogenous Ig lambdaL and kappaL chain gene loci cloned from peripheral blood lymphocytes of an NBS patient and of healthy individuals. These findings suggest that the Nbs1/Mre11/Rad50 complex is not involved in coding end processing of V(D)J recombination.
Mol Immunol 2000 Oct
PMID:Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome. 1128 95

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.
Mol Cell Biol 2001 Aug
PMID:Chk2 activation dependence on Nbs1 after DNA damage. 1143 75

Repair of DNA double-strand breaks is essential for maintenance of genomic stability, and is specifically required for rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) loci during development of the immune system. Abnormalities in these repair processes also contribute to oncogenic chromosomal rearrangements that underlie many lymphoid malignancies. Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition characterized by immunodeficiency, radiation sensitivity, and increased predisposition to lymphoid cancers bearing oncogenic Ig and TCR locus translocations. NBS patients fail to produce nibrin, a protein required for the nuclear localization and function of a DNA repair complex that includes Mre11 and Rad50. Mre11 has biochemical properties that suggest a potential role in V(D)J recombination. We studied V(D)J recombination in NBS cells in vitro and in vivo, using cell lines and peripheral blood leukocyte DNA from NBS patients. We found that NBS cells were competent to rejoin signal substrates with normal efficiency and high fidelity. Coding substrates were similarly rejoined efficiently, and coding end structures appeared normal. In B cells from NBS patients, the spectrums of IgH CDR3 regions were diverse and normally distributed. Moreover, the lengths and composition of Igkappa VJ joins and IgH VDJ joins derived from NBS and normal subjects were indistinguishable. Our data indicate that nibrin plays no essential role in V(D)J recombination and is not required for the generation of an apparently diverse B cell repertoire.
Mol Immunol 2000 Dec
PMID:V(D)J rearrangement in Nijmegen breakage syndrome. 1145 18

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.
Mol Microbiol 2001 Aug
PMID:Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease. 1153 50

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.
Mol Cell 2001 Nov
PMID:Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. 1174 45

The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways. To find out what the common function of R/M in these pathways might be, we investigated its architecture. Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members. R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain. DNA end-bound R/M oligomers could tether linear DNA molecules. These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.
Mol Cell 2001 Nov
PMID:Human Rad50/Mre11 is a flexible complex that can tether DNA ends. 1174 47

The Mre11 complex is a multisubunit nuclease that is composed of Mre11, Rad50 and Nbs1/Xrs2. Mutations in the genes that encode components of this complex result in DNA- damage sensitivity, genomic instability, telomere shortening and aberrant meiosis. The molecular defect that underlies these phenotypes has long been thought to be related to a DNA repair deficiency. However, recent studies have uncovered functions for the Mre11 complex in checkpoint signalling and DNA replication.
Nat Rev Mol Cell Biol 2002 May
PMID:The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. 1198 66

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
Microbiol Mol Biol Rev 2002 Dec
PMID:Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. 1245 86


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