Measurement of trinucleotide repeat instability in yeast

Abstract

Instability of trinucleotide repeats (TNRs) is responsible for at least 15 hereditary disorders in humans. There is substantial evidence that alterations of the number of these repeats are due to the formation of secondary structures such as hairpins or slipped intermediates during lagging-strand DNA replication. Saccharomyces cerevisiae (S. cerevisiae) Dna2, which possesses endonuclease and helicase activities, plays a critical role in Okazaki fragment processing. The N-terminal 45-kDa domain of Dna2 binds to hairpin structures, targeting the enzyme specifically to a secondary-structured flap DNA. This Dna2-associated activity is required for the endonucleolytic removal of hairpin flaps by Dna2 with the aid of its helicase activity. Thus, the efficient removal of flaps, generated during the replication of lagging strand templates containing TNRs, requires the coordinated action of all three activities of Dna2. Here we show in vivo that Dna2 endonuclease/helicase is required for the stable maintenance of TNRs in S. cerevisiae. Our results demonstrate that the malfunction of Dna2 during Okazaki fragment processing in eukaryotes contributes to TNR instability.