Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Yeast system to study expansions of DNA repeats
Expansions of tandem DNA repeats, which are responsible for numerous hereditary disorders in humans, are often large-scale events wherein multiple repeat units are acquired in a single step. Our studies were concentrated on the mechanisms and consequences of expansions of (GAA)n repeats, which are responsible for the disease, Friedreich?s ataxia. We have developed a unique experimental system to analyze large-scale repeat expansions in yeast, which allowed us to monitor expansions of the premutation range (78-to-150 copies) of (GAA)n repeats well into the disease range (200-to-450 copies). Fig.1A shows our system, based on the URA3 reporter split by an actin intron carrying various number of (GAA)n repeats. These cassettes were integrated into chromosome III in two orientations relative to the ARS306 replication origin. Large-scale expansions of GAA repeats led to the reporter?s inactivation allowing expanded clones to grow in the presence of 5-FOA (Fig.1B). Remarkably, the rates of expansion events per replication in our system increased exponentially with the repeat?s length (Fig.1C), which is quite similar to what was observed in human pedigrees suggesting that mechanisms of repeat expansions are similar for all eukaryotes.
The analysis of the lengths of expansions in the case of (GAA) 150 revealed the selection cutoff of the experimental system to be 170-180 repeats (Fig.1D). For 150 copies of the GAA repeat, we detected a normal length distribution of the expanded repeats with a mean length of 220 copies, which is significantly longer than the selection threshold (Fig 1D). You can see from Fig.1D, that expansions are large-scale in their nature. These observations gave us the existence of a preferential expansion increment corresponding to approximately 1.5-times of the repeat?s length. The existence of this bias in the expansion size explains the dramatic (three orders of magnitude) difference in the expansion rates between the shortest and longest GAA repeats presented in Fig.1C, as more than one expansion step would be necessary to reach the selection cutoff for the shorter repeats. To gain a better insight into the mechanisms of GAA repeat instability, we conducted a preliminary screen for mutants in various aspects of DNA metabolism, such as DNA replication, repair and recombination, which could affect the expansion rates in our system. The rate of expansions was elevated four- to six-fold in the Tof1 or Csm3 knockouts and decreased three- to four-fold in the Sgs1, Rad5 or Rad6 knockouts. Knockouts of the Rad50, Rad51, Rad52, Srs2, Rrm3, Pif1, Rad26, Msh2, Mus81 genes had little, if any, effect on the expansion rate. Since all the proteins that had a significant effect on expansion rate play a role in the replication fork stabilization, stalling and restart, we believe that expansion happens either in front or immediately behind the replication fork. Based on these data, we propose a new model for large-scale repeat expansions based on the template switching during the replication fork progression through repetitive DNA.
Also, this system allows us to monitor large-scale contractions of the expanded repeats, since those contractions should restore the functionality of the URA3 cassette, making cells URA+. The rates of such contractions are roughly 10-4 per replication, corresponding to a mutation frequency of 0.1%.
Alexander A. Shishkin1
1Department of Biology
Figure 1: A. Scheme of genetic cassette located on chromosome III. The ACT1 intron, carrying the GAA/TTC repetitive tract, was inserted into the StuI site of the URA3 gene. B. Large-scale expansions of (GAA)n repeats. C. Dependence of rates of expansion on repeat length in log-scale. D. Distribution of expansion lengths for GAA150 repeats.