Okazaki Fragment Processing and Trinucleotide Repeat Stability in S. cerevisiae: a Critical Role of the N-terminal Domain of Dna2 in Maintenance of Trinucleotide Repeats

초록

Instability of trinucleotide repeats (TNR) is responsible for at least 15 hereditary neurological disorders in humans. It is hypothesized that the disease-associated TNRs become unstable during lagging strand synthesis; when the 5'-end TNR region of Okazaki fragment dissociates from the template DNA, secondary structure can form either on the template or on the daughter strand. Resumption of DNA synthesis leads to the repeats expansion or contraction, respectively. Here, we show that dysfunction of Dna2 during processing of Okazaki fragments in S. cerevisiae causes severe instability of CTG/CAG repeats. Both endonuclease and helicase domains of Dna2 were essential, whereas the N-terminal 405 amino acid residues was dispensible for viability. Although Dna2delta405N lacking the N-terminal domain contained wild-like enzymatic activities, it was required for optimal growth indicating that it have a nonessential, but critical under some situation, role during Okazaki fragment processing. Biochemical analyses of the mutant protein suggest that the N-terminal domain functions to target Dna2 to flaps containing secondary structure. Coordinated action of three domains of Dna2 was critical to resolve and process the secondary structure developed on flaps. Consistent with these, the dna2delta05N mutant resulted in severe instability of CTG/CAG repeats and the instability was orientation-dependent. Thus, Dna2 contributes to the maintenance of TNR stability by actively removing the secondary-structured flaps.