Orientation Dependence of Trinucleotide CAG Repeat Instability in Saccharomyces cerevisiae

Abstract

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3 end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 ؋ 10 ؊2 changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5 to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5 end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.Expansions of repetitions of the trinucleotide CAG are the cause of a number of human inherited, dominant neurological and neuromuscular diseases, including Huntington's disease (14), two forms of spinocerebellar ataxia (type 1 and MachadoJoseph disease) (17,20), and myotonic dystrophy (3,7,19). Although CAG trinucleotide repetitions are present in normal alleles of the genes giving rise to these disorders, mutant alleles have tracts which are longer than those within the normal range. The long tracts within disease alleles are unstable in that children often inherit a repeat length different from that found in their affected parent. The instability most likely reflects replicative errors which occur either during the meiotic division of gametogenesis or during the mitotic divisions preceding it.The underlying cause of the instability is thought to reflect the ability of CAG repeats to form palindrome-like structures (8,22). Such structures may present problems to the replication fork as it passes through them. One study using small CAG repeats embedded in palindromes carried on phage lambda showed that they were inhibitory to plaque formation (6). Studies with Escherichia coli have also shown that CAG repeats undergo both contractions and expansions when propagated in a bacterial plasmid (16).We decided to examine the stability of CAG repeats in Saccharomyces cerevisiae because the chromatin structure and chromosomal replication of this simple eukaryote have many similarities to the chromosomal mechanics of more complex euka...

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

“…Furthermore, no rearrangements were observed on analyzing individual colonies obtained from transforming the parental plasmids into E. coli HB101. Likewise, rearrangements were not observed previously in other prior instability studies with triplet repeat inserts in E. coli HB101 (10,16,18,19,39,86,(91)(92)(93). This confirms that the rearrangements were because of an intrinsic property of the pcDNA3.1 shuttle vector that carries the SV40 origin of replication.…”

“…In case of the tetranucleotide repeats, however, instability refers to both expansions and deletions. Thus, upon initial consideration, our results appear to be similar to the results with the triplet repeat sequences, including CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC, in which orientation II was shown to be more unstable (9,10,16,18,19,21,22,39,52,85,86,95,96). However, the CCTG sequences are genetically unstable in the orientation prone to expand (orientation II) (Fig.…”

“…Previous studies showed a marked effect of orientation of trinucleotide repeats (CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC) with respect to the origin of replication (10,16,18,19,21,39,85,86). Therefore, we studied plasmids carrying the (CCTG⅐CAGG) repeats in both orientations.…”

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

“…The disruption of the RAD27 gene of Saccharomyces cerevisiae (a homologue of human flap endonuclease 1, FEN-1) did affect the instability of TNR while the disruption of mismatch or recombination related genes did not change the rates of TNR instability (Maurer et al,1996;Freudenreich et al, 1998). Zakian et al showed that a deletion mutant of RAD27 caused lengthdependent destabilization of CTG tracts and increased in its expansion frequency.…”

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

“…DNA replication (5)(6)(7)(8) and repair including methyl-directed mismatch repair (9 -11), nucleotide excision repair (12), DNA polymerase III exonucleolytic proofreading (13), and double-strand break repair (14) have been implicated.…”