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J. Biol. Chem., Vol. 278, Issue 46, 45171-45181, November 14, 2003

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Regulation of Mre11/Rad50 by Nbs1

EFFECTS ON NUCLEOTIDE-DEPENDENT DNA BINDING AND ASSOCIATION WITH ATAXIA-TELANGIECTASIA-LIKE DISORDER MUTANT COMPLEXES^*

Ji-Hoon Lee, Rodolfo Ghirlando, Venugopal Bhaskara¶, Michaela R. Hoffmeyer||, Jian Gu||, and Tanya T. Paull||**

From the Department of Molecular Genetics and Microbiology, the ^||Institute of Cellular and Molecular Biology, and the ^¶Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712 and the NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892

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 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.

Received for publication, August 6, 2003 , and in revised form, September 3, 2003.

^* This work was supported by the Kimmel Cancer Foundation and by National Institutes of Health Grant CA940008-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Molecular Genetics and Microbiology, University of Texas, 1 University Station, A4800, Austin, TX 78712. Tel.: 512-232-7802; Fax: 512-232-3432; E-mail: tpaull{at}icmb.utexas.edu.

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