Role of DNA Polymerases in Repeat-Mediated Genome Instability

Abstract

SUMMARY Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAA)n repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

Genome rearrangements caused by interstitial telomeric sequences in yeast

“…Because long (>1-kb) introns are inefficiently removed from pre-mRNA in yeast (12), large-scale expansions of the (GAA) n tracts produce Ura − clones that might be selected on the medium containing 5-fluoroorotic acid (5-FOA) (11). 5-FOA-resistant clones also resulted from large deletions around those repeats (11,13), as well as from the repeatinduced mutagenesis in the body of the URA3 gene (13)(14)(15).…”

“…In the control strain (SMY803) with no Ytel sequences in the reporter, the rate of 5-FOA R derivatives was about 0.4 × 10 −7 per division. This rate was elevated in strains with (TGTGTGGG) 8 and (TGTGTGGG) 15 YTel tracts 20-fold and 125-fold, respectively ( Table 1). The 5-FOA R derivatives of all three strains initially were examined by PCR analysis with the primers (UIRL1/UIRL2) that flanked the YTel tracts (Fig.…”

“…either the (TGTGTGGG) 8 or the (TGTGTGGG) 15 insertions (strains SMY752 and SMY749, respectively) were Ura + and 5-FOA S , although a quantitative measurement of gene expression (Fig. S2) indicated that the (TGTGTGGG) 15 Ytel tract reduced expression of the spliced URA3 transcript.…”

“…S2) indicated that the (TGTGTGGG) 15 Ytel tract reduced expression of the spliced URA3 transcript. Because telomeric tracts in the opposite orientation resulted in an even stronger suppression of splicing, we did not attempt a direct comparison of the effects of tract orientation on genome stability.…”

“…Because small changes in the number of repeats did not generate a 5-FOA R strain, we also looked for small expansions or contractions of the (TGTGTGGG) 15 tract in the nonselective conditions. To this end, 20 clones each from 12 independent cultures grown nonselectively were analyzed by PCR.…”