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J. Biol. Chem., Vol. 280, Issue 39, 33311-33317, September 30, 2005

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Srs2 Helicase of Saccharomyces cerevisiae Selectively Unwinds Triplet Repeat DNA^*

From the Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805

Trinucleotide repeat expansions are the mutational cause of at least 15 genetic diseases. In vitro, single-stranded triplet repeat DNA forms highly stable hairpins, depending on repeat sequence, and a strong correlation exists between hairpin-forming ability and the risk of expansion in vivo. Hairpins are viewed, therefore, as likely mutagenic precursors to expansions. If a helicase unwinds the hairpin, it would be less likely to expand. Previous work indicated that yeast Srs2 DNA helicase selectively blocks expansions in vivo (Bhattacharyya, S., and Lahue, R. S. (2004) Mol. Cell. Biol. 24, 7324–7330). For example, srs2 mutants, including an ATPase-defective point mutant, exhibit substantially higher expansion rates than wild type controls. In contrast, mutation of another helicase gene, SGS1, had little effect on expansion rates. These findings prompted the idea that Srs2 might selectively unwind triplet repeat hairpins. In this study, DNA helicase assays were performed with purified Srs2, Sgs1, and Escherichia coli UvrD (DNA helicase II). Srs2 shows substantially faster unwinding than Sgs1 or UvrD on partial duplex substrates containing (CTG)·(CTG) sequences, provided that Srs2 encounters the triplet repeat DNA immediately on entering the duplex. Srs2 was also faster at unwinding (CAG)·(CAG)- and (CCG)·(CCG)-containing substrates and an intramolecular (CTG)·(CTG) hairpin. In contrast, all three enzymes unwind about equally well control substrates with either Watson-Crick base pairs or mismatched substrates with non-CNG repeats. Overall, the selective unwinding activity of Srs2 on triplet repeat hairpin DNA helps explain the genetic evidence that Srs2, not the RecQ homolog Sgs1, is a preferred helicase for preventing expansions.

Received for publication, March 25, 2005 , and in revised form, August 3, 2005.

^* This work was supported by National Institutes of Health Grant R01 GM GM61961 (to R. S. L.), by National Institutes of Health Training Grant T32 CA09746 (to S. B.), and by NCI, National Institutes of Health Cancer Center Support Grant P30 CA36727 (to the Eppley Institute). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental graphs of the data for Figs. 5, 6, 7, 8, 9.

¹ To whom correspondence should be addressed: Eppley Institute for Research in Cancer and Allied Diseases, Box 986805, University of Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-4619; Fax: 402-559-8270; E-mail: rlahue{at}unmc.edu.

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