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J. Biol. Chem., Vol. 283, Issue 35, 23964-23971, August 29, 2008

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Physiological and Biochemical Defects in Carboxyl-terminal Mutants of Mitochondrial DNA Helicase^*

Yuichi Matsushima, Carol L. Farr, Li Fan, and Laurie S. Kaguni¹

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.

Received for publication, May 13, 2008 , and in revised form, June 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM45295 (to L. S. K.) and RO1 CA112093 and PO1 CA92584 (NCI; to John A. Tainer in support of L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Michigan State University, East Lansing MI 48824-1319. Tel.: 517-353-6703; Fax: 517-353-9334; E-mail: lskaguni{at}msu.edu.

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