title: Catalytic mechanism of bacteriophage T4 Rad50 ATP hydrolysis.
abstract: Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Herdendorf
authors.forename: Scott W
authors.initials: SW
authors.lastname: Nelson
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014783:Viscosity
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D003851:Deoxyribonucleases; D000251:Adenosine Triphosphatases