Structural and functional analysis of Mre11-3.
The Mre11, Rad50 and Nbs1 proteins make up the conserved multi-functional Mre11 (MRN) complex involved in multiple, critical DNA metabolic processes including double-strand break repair and telomere maintenance. The Mre11 protein is a nuclease with broad substrate recognition, but MRN-dependent processes requiring the nuclease activity are not clearly defined. Here, we report the functional and structural characterization of a nuclease-deficient Mre11 protein termed mre11-3. Importantly, the hmre11-3 protein has wild-type ability to bind DNA, Rad50 and Nbs1; however, nuclease activity was completely abrogated. When expressed in cell lines from patients with ataxia telangiectasia-like disorder (ATLD), hmre11-3 restored the formation of ionizing radiation-induced foci. Consistent with the biochemical results, the 2.3 A crystal structure of mre11-3 from Pyrococcus furiosus revealed an active site structure with a wild-type-like metal-binding environment. The structural analysis of the H85L mutation provides a detailed molecular basis for the ability of mre11-3 to bind but not hydrolyze DNA. Together, these results establish that the mre11-3 protein provides an excellent system for dissecting nuclease-dependent and independent functions of the Mre11 complex.
Arthur
L Matthew
LM
Radiation Oncology Research Laboratory, Department of Radiation Oncology, Molecular and Cell Biology Graduate Program and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Gustausson
Karin
K
Hopfner
Karl-Peter
KP
Carson
Christian T
CT
Stracker
Travis H
TH
Karcher
Annette
A
Felton
Diana
D
Weitzman
Matthew D
MD
Tainer
John
J
Carney
James P
JP
D017766:Acid Anhydride Hydrolases; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D000076228:MRE11 Homologue Protein; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D004247:DNA; D005092:Exonucleases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes