Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae

Abstract

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-f luoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 ؋ 10 ؊5 to 4 ؋ 10 ؊5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500-to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientationdependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

“…6B). When wild type protein was used, the FEN cleavage products (17,16, and 15 nt) can be observed, followed by EXO cleavage products (10 -14 nt), which increase in intensity as the reaction progresses. When E176A is used, only FEN cleavage products are observed, along with a low fraction of EXO cleavage products.…”

“…4). Repeats that form DNA secondary structures are more likely to undergo TNR expansion (16), because these structures inhibit recognition and processing by the repair machinery (46,47 20 has been demonstrated by NMR to have stable secondary structure in solution (6). Here we employ the same (CTG) 20 hairpin substrate labeled at 5Ј-and 3Ј-ends to compare the cleavage efficiencies of WT and mutant proteins.…”

“…Thus, multiple structures may play a role in genomic instability. The location of the secondary structure with respect to leading/lagging and nascent/template strands is also important in determining the repeat stability (15,16). In vivo studies of TNR stability have taken advantage of model systems in Escherichia coli, yeast, mice, and cell lines.…”

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…the direction of replication) with respect to the types and amount of products (10,16,18,22,52,85,95,96). To determine whether a similar orientation effect was observed for the tetranucleotide repeats, (CCTG⅐CAGG) n repeats were cloned in both orientations with FIG.…”

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

“…Bacterial (Escherichia coli) 19,20 and yeast (Saccharomyces cerevisiae) [21][22][23][24] models support a role for DNA replication in TNR instability in that the direction of replication through the TNR tract affects its stability. In bacterial and yeast systems, repeat deletions predominate regardless of the direction of replication, but the frequency of deletions is higher when the CTG strand, rather than the CAG strand, is the template for lagging-strand synthesis 19 .…”