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

被引:82
作者
Lee, JH
Ghirlando, R
Bhaskara, V
Hoffmeyer, MR
Gu, J
Paull, TT
机构
[1] Univ Texas, Dept Mol Genet & Microbiol, Austin, TX 78712 USA
[2] Univ Texas, Inst Cellular & Mol Biol, Austin, TX 78712 USA
[3] Univ Texas, Dept Chem & Biochem, Austin, TX 78712 USA
[4] NIDDK, NIH, Dept Hlth & Human Serv, Bethesda, MD 20892 USA
关键词
D O I
10.1074/jbc.M308705200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.
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收藏
页码:45171 / 45181
页数:11
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