Large-scale expansions of Friedreich's ataxia GAA repeats in yeast

Abstract

Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats responsible for the human disease Friedreich's ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeat's orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

Fig. 1. Selectable system to detect large-scale GAA repeats expansions in yeast

A. Selectable cassettes for repeat expansions in two orientations in chromosome III. Gray areas correspond to split URA3 gene, hatched areas – ACT1 intron, white rectangle – GAA repeat. B. Characteristic results of the PCR analysis of 5-FOA^R clones originated from a cassette with 100 GAA repeats, showing expansions, mutations and deletions. Black arrow designates electrophoretic mobility of the PCR-product from the original (GAA)₁₀₀ repeat. C. Schematic representation of mutations and small-scale deletions in 5-FOA^R clones. Stars correspond to point mutations, triangle shows a microinsertion, small deletions are marked by gapped lines with sequence microhomologies in parentheses. D. Mapping of large deletions using Southern blot hybridization.

Fig. 2. Rates and length distributions of expanded repeats

A. Rates and 95% confidence intervals of expansions, mutations and deletions for the GAA repeats of varying lengths. B. Dependence of expansion rates on repeat lengths. Red diamonds – expansions, blue circles – mutations, green triangles - large deletions. C. Length distributions of expanded repeats among 5-FOA^R clones. Black bars – expansions of the (GAA)₁₀₀ repeat; orange bars – expansions of the (GAA)₁₅₀ repeat.

Fig. 3. Expansion rates and length distributions of expanded repeats depends on balancing the intron’s length

A. Repeat expansion rates in length-balanced or unbalanced introns. B. Length distributions of expanded repeats among 5-FOA^R clones. Black bars – expansions of the (GAA)₁₀₀ repeat in the original intron; gray bars – expansions of the (GAA)₁₀₀ repeat in the length-balanced intron.

Fig. 4. Replication stalling and expansions of GAA repeats

A. Replication fork progression through the GAA100 repeat in chromosome III. Repeat orientations correspond to that in Fig. 1A. B. Rates of expansions for GAA repeats in various orientations within the replicon.

Fig. 5. Effects of expanded GAA repeats on the expression of the intronated URA3 gene

A. Diagram showing primers used for the RT-PCR experiments: black arrows – for mRNA, gray arrows – for unspliced RNA. B. Characteristic results of the RT-PCR analysis of the spliced and upsliced URA3 RNA, as well as ACT1 mRNA used as normalization control. RT- is a negative control without reverse transcriptase, to rule out DNA contamination. C. Quantitative analysis of the data in (B). Black bars – normalized spliced RNA, hatched bars – normalized unspliced RNA.

Fig. 6. Proposed mechanisms of expansions of the GAA repeats (see text for details)

The homopurine strand of the repeat is shown in red, while homopyrimidine strand is in green. Blue hexameric ring represents the Mcm2-7 replicative DNA helicase, purple star corresponds to the fork pausing complex Tof1/Csm3/Mrc1, yellow circles show leading and lagging DNA polymerases, gray square represents Rad5 and, possibly, Sgs1 implicated in DNA template switching.