Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes. 1296 88

End joining of double-strand breaks (DSBs) requires Ku proteins and frequently involves base pairing between complementary terminal sequences. To define the role of terminal base pairing in end joining, two oppositely oriented HO endonuclease cleavage sites separated by 2.0 kb were integrated into yeast chromosome III, where constitutive expression of HO endonuclease creates two simultaneous DSBs with no complementary end sequence. Lack of complementary sequence in their 3' single-strand overhangs facilitates efficient repair events distinctly different from when the 3' ends have a 4-bp sequence base paired in various ways to create 2- to 3-bp insertions. Repair of noncomplementary ends results in a set of nonrandom deletions of up to 302 bp, annealed by imperfect microhomology of about 8 to 10 bp at the junctions. This microhomology-mediated end joining (MMEJ) is Ku independent, but strongly dependent on Mre11, Rad50, and Rad1 proteins and partially dependent on Dnl4 protein. The MMEJ also occurs when Rad52 is absent, but the extent of deletions becomes more limited. The increased gamma ray sensitivity of rad1Delta rad52Delta yku70Delta strains compared to rad52Delta yku70Delta strains suggests that MMEJ also contributes to the repair of DSBs induced by ionizing radiation.
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PMID:Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. 1461 21

The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.
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PMID:Microhomology-dependent end joining and repair of transposon-induced DNA hairpins by host factors in Saccharomyces cerevisiae. 1472 78

Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the 'genomic context' combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK-HerA superfamily ATPases.
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PMID:Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging. 1546 93

The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.
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PMID:Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast. 1554 77

When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends.
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PMID:The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends. 1616 95

Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.
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PMID:Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation. 1687 33

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.
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PMID:The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity. 1769 79

Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.
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PMID:Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. 1804 58

NurA is a novel 5'-3' exonuclease that is closely linked to Mre11 and Rad50 homologues in most thermophilic archaea. We report a physical and functional interaction between NurA (StoNurA) and single-stranded DNA-binding protein (StoSSB) from the hyperthermophilic archaeon Sulfolobus tokodaii. StoSSB was identified as a novel StoNurA-interacting protein by pull-down assay using Ni-NTA agarose beads and MALDI-TOF mass spectrometry. The direct interaction between StoNurA and StoSSB was further confirmed by yeast two-hybrid and co-immunoprecipitation analysis. The interaction was supposed to have functional significance because it was found that StoSSB inhibited the 5'-3' ssDNA and dsDNA exonuclease and ssDNA endonuclease activities of StoNurA. Our results suggest that NurA may function closely together with SSB in DNA transactions in archaea.
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PMID:Physical and functional interaction between archaeal single-stranded DNA-binding protein and the 5'-3' nuclease NurA. 1819 1


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