Work

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Year: 2015

Type: article

Abstract: Background: The Mre11-Rad50 (MR) complex coordinates DNA double strand break repair.Results: Mutations of the Rad50 coiled-coil disrupt the enzymatic functions of the MR complex. Conclusion: The Rad50 coiled-coil regulates and mediates communication between the enzymatic domains of Rad50 and Mre11.Significance: The Rad50 coiled-coil plays a more direct role in the enzymatic function of Rad50 and Mre11 than was previously assumed.Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks.Rad50 contains an antiparallel 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.

Source: Journal of Biological Chemistry

DOI: 10.1074/jbc.m115.675132