Dimerization of the Rad50 protein is independent of the conserved hook domain.
The Mre11 complex (Mre11-Rad50-Nbs1) is involved in a diverse array of DNA metabolic processes including the response to DNA double-strand breaks (DSBs). The structure of Rad50 plays a key role in the DNA-binding and end-bridging activity of the complex. An interesting feature within the central portion of the Rad50 protein is the Rad50 hook region that is defined by the highly conserved CXXC motif. The structure of the Pyrococcus furiosus Rad50 hook region revealed an intermolecular dimerization of Rad50 through the coordination of a zinc ion by the four cysteines. Biochemical and genetic analysis in Saccharomyces cerevisiae have shown that mutations in the conserved cysteines impact all functions of the Mre11 complex including interaction with Mre11, increased sensitivity to DSB inducing agents, telomere maintenance and intrachromosomal association. Mutations in the yeast hook domain can lead to increased chromosome fragmentation, suggesting that the hook domain of Rad50 is essential for the tethering of chromosome ends. In this study, we have examined the effects of mutating the key cysteine residues in the hook domain of human Rad50 (hRad50), focusing on the interactions Rad50 has with itself, Mre11 and DNA. Our results reveal that mutation of the conserved cysteine residues abrogates dimerization at the hook domain in hRad50; however, disrupting dimerization at this domain does not appear to impair the interaction of full-length hRad50 with itself and hMre11 or affect DNA-binding activity of the hMre11-Rad50 complex.
Cahill
Dana
D
The Radiation Oncology Research Laboratory and The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Carney
James P
JP
D017766:Acid Anhydride Hydrolases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D000076228:MRE11 Homologue Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D000076228:MRE11 Homologue Protein; C415984:Rad50 protein, archaeal; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes; D003545:Cysteine