Structure of the Rad50·Mre11 DNA Repair Complex fromSaccharomyces cerevisiae by Electron Microscopy

Anderson, David E.; Trujillo, Kelly M.; Sung, Patrick; Erickson, Harold P.

doi:10.1074/jbc.m106179200

Cited by 103 publications

(67 citation statements)

References 32 publications

(57 reference statements)

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“…Also unlike Ku, MRX plays a role in HR, possibly regulating repair pathway utilization through actions in 59 resection of DSB ends (Connelly and Leach 2002;Symington 2002). Rad50 is composed of two globular ATPase domains separated by a long coiled-coil region that self-associates at its end (Anderson et al 2001;Wiltzius et al 2005). Mre11 has an N-terminal nuclease domain, which binds near the Rad50 ATPase to create a DNA-binding head (Usui et al 1998;Hopfner et al 2001).…”

Section: N Onhomologous End Joining (Nhej) Is a Prin-mentioning

confidence: 99%

Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

2008

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Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.

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Section: N Onhomologous End Joining (Nhej) Is a Prin-mentioning

confidence: 99%

Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

2008

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“…Subsequent studies revealed similar defects in meiosis, repair of IR-induced DNA damage, telomere length, nonhomologous end joining (NHEJ), and the intra-S phase checkpoint in mre11, rad50, and xrs2 null mutants (Haber 1998;D'Amours and Jackson 2002). The corresponding proteins form a high-affinity complex with an 2:2:1 stoichiometry of Mre11, Rad50, and Xrs2 (Anderson et al 2001;Chen et al 2001). Both Mre11 and Rad50 are conserved and are homologous to the Escherichia coli SbcD and SbcC proteins, respectively (Sharples and Leach 1995).…”

mentioning

confidence: 99%

Mutations in Mre11 Phosphoesterase Motif I That Impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 Complex Stability in Addition to Nuclease Activity

Krogh¹,

Llorente²,

Lam

et al. 2005

Genetics

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The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited ,2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.

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“…28 In response to DNA damage, 53BP1 becomes hyperphosphorylated by ATM and binds to chromatin, forming discrete nuclear foci that colocalize with MRE11. [29][30][31][32][33] 53BP1 is also required for at least a subset of ataxia telangiectasia-mutated (ATM)-dependent phosphorylation events at sites of DNA breaks 34 and has recently been implicated in a novel ATM-dependent pathway, involving Artemis, required for the processing of a subset of DSBs prior to rejoining by nonhomologous end-joining (NHEJ). 35,36 53BP1 can bind both ss and ds DNA with high affinity, thus possibly acting as an alignment factor for DSBs.…”

Section: Introductionmentioning

confidence: 99%

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

Boisvert¹,

Rhie²,

Richard³

et al. 2005

Cell Cycle

124

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The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore-binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylargininespecific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few γ-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and as the site of methylation by PRMT1.

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Structure of the Rad50·Mre11 DNA Repair Complex fromSaccharomyces cerevisiae by Electron Microscopy

Cited by 103 publications

References 32 publications

Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

Mutations in Mre11 Phosphoesterase Motif I That Impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 Complex Stability in Addition to Nuclease Activity

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

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