title: Structural and functional analysis of Mre11-3.
abstract: 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.
authors.affiliation: 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.
authors.forename: L Matthew
authors.initials: LM
authors.lastname: Arthur
authors.forename: Karin
authors.initials: K
authors.lastname: Gustausson
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Hopfner
authors.forename: Christian T
authors.initials: CT
authors.lastname: Carson
authors.forename: Travis H
authors.initials: TH
authors.lastname: Stracker
authors.forename: Annette
authors.initials: A
authors.lastname: Karcher
authors.forename: Diana
authors.initials: D
authors.lastname: Felton
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Weitzman
authors.forename: John
authors.initials: J
authors.lastname: Tainer
authors.forename: James P
authors.initials: JP
authors.lastname: Carney
mesh_terms: 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
chemical_list: 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