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Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron–sulfur cluster domain

Pokharel, Subhash and Campbell, Judith L. (2012) Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron–sulfur cluster domain. Nucleic Acids Research, 40 (16). pp. 7821-7830. ISSN 0305-1048. doi:10.1093/nar/gks534.

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Dna2 nuclease/helicase is a multitasking protein involved in DNA replication and recombinational repair, and it is important for preservation of genomic stability. Yeast Dna2 protein contains a conserved putative Fe–S (iron–sulfur) cluster signature motif spanning the nuclease active site. We show that this motif is indeed an Fe–S cluster domain. Mutation of cysteines involved in metal coordination greatly reduces not just the nuclease activity but also the ATPase activity of Dna2, suggesting that the nuclease and helicase activities are coupled. The affinity for DNA is not significantly reduced, but binding mode in the C to A mutants is altered. Remarkably, a point mutation (P504S), proximal to the Fe–S cluster domain, which renders cells temperature sensitive, closely mimics the global defects of the Fe–S cluster mutation itself. This points to an important role of this conserved proline residue in stabilizing the Fe–S cluster. The C to A mutants are deficient in DNA replication and repair in vivo, and, strikingly, the degree to which they are defective correlates directly with degree of loss of enzymatic activity. Taken together with previous results showing that mutations in the ATP domain affect nuclease function, our results provide a new mechanistic paradigm for coupling between nuclease and helicase modules fused in the same polypeptide.

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Additional Information:© 2012 The Author(s). Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received April 5, 2012; Revised May 10, 2012; Accepted May 11, 2012; Published online 7 June 2012. The authors thank Robert Bambara, University of Rochester, and members of the Campbell laboratory for their discussions and comments on the manuscript. Funding: National Institutes of Health [GM100196]; Congressionally Directed Medical Research Programs [W81XWH-09-1-0041]; Ellison Medical Foundation [AG-SS-2143-08]. Funding for open access charge: California Institute of Technology.
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Congressionally Directed Medical Research ProgramsW81XWH-09-1-0041
Ellison Medical FoundationAG-SS-2143-08
Issue or Number:16
Record Number:CaltechAUTHORS:20121026-135506376
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Official Citation: Subhash Pokharel and Judith L. Campbell Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron–sulfur cluster domain Nucl. Acids Res. (2012) 40(16): 7821-7830 first published online June 7, 2012 doi:10.1093/nar/gks534
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:35127
Deposited By: Tony Diaz
Deposited On:30 Oct 2012 23:59
Last Modified:09 Nov 2021 23:12

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