Abstract
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) x (CTG) sequences, provided that Srs2 encounters the triplet repeat DNA immediately on entering the duplex. Srs2 was also faster at unwinding (CAG) x (CAG)- and (CCG) x (CCG)-containing substrates and an intramolecular (CTG) x (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.
Highlights
These results suggested that Srs2 helps prevent triplet repeat expansions by unwinding hairpin intermediates and that Srs2 is effective at unwinding triplet repeat DNA
This study demonstrates that purified Srs2 is substantially more active (ϳ3-fold) at unwinding CNG repeat substrates compared with two other helicases
The selectivity of Srs2 helicase activity in vitro correlates with its signature mutator phenotype in vivo, where only CNG repeat expansion rates are elevated in srs2 mutants [29]
Summary
Our in vitro results reported here indicate that Srs2 is substantially more active on CNG repeat DNA than Sgs1 and UvrD, largely because of the ability of Srs2 to unwind triplet repeat structures encountered immediately when the enzyme enters the duplex. In contrast to the earlier finding (Fig. 3A) that Srs2 was somewhat slower on control DNA, the results in Fig. 3B support the idea that Srs2 at equimolar concentration is more active than UvrD or Sgs1 at unwinding CTG repeat-containing DNA duplexes.
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