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, 19 (7), 4766-73

Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces Cerevisiae

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Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces Cerevisiae

B D Harfe et al. Mol Cell Biol.

Abstract

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.

Figures

FIG. 1
FIG. 1
Sequence spectra of +1 frameshift events in wild-type and MMR-defective strains. The sequences of the entire +1 and −1 assay reversion windows are shown; nucleotides are numbered from the XbaI site upstream of the LYS2 gene. Nt 746 was deleted, and 3 nt were changed (A767C, T781C, and T753C, lowercase letters; see Materials and Methods) to create the +1 assay strain. The −1 assay strain was created by filling in a BglII site (added nucleotides are underlined and in boldface; 8). Dashes denote sequences that are present in the −1 assay system but are absent in the +1 reversion window. The −1 mutation spectrum was adopted from Greene and Jinks-Robertson (8). The locations of single base pair insertions (+) and deletions (Δ) are indicated. The locations of deletion events that occurred between two perfect direct repeats are indicated by the abbreviation del. One copy of the 10-bp direct repeat at the endpoints of the 94-bp deletion is boxed; the second 10-bp direct repeat lies 5′ of the reversion window and is not shown. The deletion extending from T785 to T810 has 4-bp direct repeats (TTTG) at its ends; the G of the second repeat is outside of the reversion window. cIns and cDel denote the locations of complex insertion and deletion events, respectively. The complex events in the lys2ΔA746 reversion spectrum in wild-type cells were as follows (underlining indicates the positions of base substitutions; asterisks indicate the positions of the inserted or deleted bases; base changes are in uppercase): cIns1, t*gttccgtttggc changed to tAgttccgtttgTc; cIns2, tttc*aaa changed to tttAAaaa; cIns3, ga*tttcaaattg changed to gaAtttAaaaAtg; cIns4, aaaaaa*ttc changed to aaaaaaAAtc; cIns5, tttttgg*aaa changed to tttttAgAaaa; cDel1, gagctcaag changed to ga*TCc*ag; cDel2, tctggaaa changed to tTt**aaa. The complex events in the msh3Δ spectrum were as follows: cIns1, ccg*******tttggcc changed to ccgCATAAGGtttgTcc; cIns2, ccggttt*gcc changed to ccTgtttTTcc.
FIG. 2
FIG. 2
Distributions of frameshift mutations in the +1 versus −1 assay systems in wild-type strains. Events were classified by location in noniterated sequences (1N) or in tandem repeated sequences (2N to 6N). Large deletions (Δ; >3 bp) and complex events were considered separate classes. The percentage of total events in each class is indicated.

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