Length and Sequence Heterozygosity Differentially Affect HRAS1 Minisatellite Stability During Meiosis in Yeast

Abstract

Minisatellites, one of the major classes of repetitive DNA sequences in eukaryotic genomes, are stable in somatic cells but destabilize during meiosis. We previously established a yeast model system by inserting the human Ha-ras/HRAS1 minisatellite into the HIS4 promoter and demonstrated that our system recapitulates all of the phenotypes associated with the human minisatellite. Here we demonstrate that meiotic minisatellite tract-length changes are half as frequent in diploid cells harboring heterozygous HRAS1 minisatellite tracts in which the two tracts differ by only two bases when compared to a strain with homozygous minisatellite tracts. Further, this decrease in alteration frequency is entirely dependent on DNA mismatch repair. In contrast, in a diploid strain containing heterozygous minisatellite tract alleles differing in length by three complete repeats, length alterations are observed at twice the frequency seen in a strain with homozygous tracts. Alterations consist of previously undetectable gene conversion events, plus nonparental length alteration events seen previously in strains with homozygous tracts. A strain containing tracts with both base and length heterozygosity exhibits the same level of alteration as a strain containing only length heterozygosity, indicating that base heterozygosity-dependent tract stabilization does not affect tract-length alterations occurring by gene conversion. M INISATELLITES are tracts of tandemly repeatedand MS32, for example) exhibit very high rates of tractlength alterations; these types are often utilized for fo-DNA in which the repeat unit ranges in length rensic DNA fingerprinting. Alterations in minisatellite from 10 to 100 nucleotides. In addition, within a given tracts are often polar in nature (Armour et al. 1993; tract, variations on the canonical repeat sequence can Jeffreys et al. 1994), occurring at one side of the tract occur. For example, the human HRAS1 minisatellite tract preferentially. This polarity, and the meiotic timing of is commonly composed of tandem iterations of four varithe alterations, has led to the hypothesis that alterations ants of a 28-bp sequence: 5Ј-ACACTCGCCCTTCTCTC in minisatellite sequences result from a recombination CAGGGGACGCC-3Ј. Repeats contain either a C or a G event at the locus rather than during replication. at the seventh and/or fifteenth nucleotide (as indicated Identification of the meiotic factors underlying miniby underlining) (Capon et al. 1983). Additionally, in satellite stability has been complicated by the high level the human population there are four different common of variability exhibited by minisatellite sequences in the alleles of the minisatellite, designated a1-a4 (Krontiris human population. We developed the yeast HIS4-HRAS1 et al. 1985), which consist of a particular combination model system to overcome these difficulties (Jauert et of the four repeat types in a defined order. Alleles are al. 2002). We replaced the Saccharomyces cerevisiae HIS4 considered common if they occur in Ͼ7% of...

Minisatellite alterations in ZRT1 mutants occur via RAD52-dependent and RAD52-independent mechanisms in quiescent stationary phase yeast cells

“…1). To construct this plasmid, a PCR product containing the HRAS1 minisatellite repeats from DTK759a [15] and Xba I-cleavable ends was amplified using primers 35138391 and 42180832. This cassette was digested with Xba I and ligated into the Xba I site of the ADE2 fragment contained in the plasmid pEAS8 [25].…”

“…Human minisatellites have been shown to change in tract length and repeat composition during meiosis, while remaining relatively stable during mitotic cell cycles [1]. We previously demonstrated that these phenotypes are recapitulated in the budding yeast Saccharomyces cerevisiae [14, 15]. Since the patterns of minisatellite alteration are similar in yeast and human cells, we used the more genetically tractable yeast to identify factors that control minisatellite stability during meiosis.…”

Heterozygosity and mutation rate: evidence for an interaction and its implications

“…A more plausible figure is suggested by the experiments in yeast, where heterozygous human minisatellites have twice the mutation rate of equivalent homozygous loci. (7,8) The situation with respect to HI SNP is more difficult to determine. In their study of substitution rates around deletions, Tian et al (70) estimated that divergence between mouse and rat drops by almost a factor of 3, from 0.141 to 0.056, when moving from 100 to 450 bases away from the deletion.…”

“…Consequently, if heterozygous sites exhibit increased mutability, larger populations will tend to have relatively higher mutation rates. This idea of heterozygote instability (HI) is treated as novel or even heretical by population geneticists and evolutionary biologists (personal observation), yet molecular studies show clearly that the requisite elements are present, shown most clearly in yeast (7,8) but with evidence of similar processes in higher organisms too. During synapsis, the stage of meiosis where homologous chromosomes pair, extensive regions of heteroduplex DNA are formed, comprising one strand from each of the two homologues.…”

“…Such a pattern is suggested by an observation on another class of tandem repeat: human minisatellites inserted into the yeast genome are twice as mutable when heterozygous as when homozygous. (7,8) Importantly, key enzymes involved in these pathways in yeast appear to be present in all higher organisms, (16,18,21) and gene conversion without exchange…”