title: Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.
abstract: Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.
authors.affiliation: From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 and.
authors.forename: Tasida
authors.initials: T
authors.lastname: Barfoot
authors.affiliation: From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 and.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Herdendorf
authors.affiliation: From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 and.
authors.forename: Bryanna R
authors.initials: BR
authors.lastname: Behning
authors.affiliation: the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Bradley A
authors.initials: BA
authors.lastname: Stohr
authors.affiliation: From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 and.
authors.forename: Yang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Kreuzer
authors.affiliation: From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 and swn@iastate.edu.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Nelson
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D005092:Exonucleases; D000251:Adenosine Triphosphatases; D015032:Zinc
keywords: ATPase; DNA repair; bacteriophage; coiled-coil; enzyme kinetics; phosphodiesterases; zinc