Maintenance of the integrity of genetic material during genome duplication requires that mutation rates be very low. The overall fidelity of DNA replication is determined at three steps: (1) the error rate of nucleotide incorporation during polymerization, (2) the efficiency with which the exonucleolytic proofreading activity of DNA polymerase removes terminal nucleotides that are incorrectly base-paired, and (3) the efficiency with which post-replicative mismatch repair (MMR) systems remove any remaining errors. Mutations that result from persistent, unrepaired replication errors generally can be classified as either insertion/deletion events involving a small number of nucleotides or base substitution events. Frameshift mutations are specifically those nucleotide insertion/deletion events that do not occur in multiples of three and consequently alter the reading frame of the translated mRNA. Whereas base substitution events often are phenotypically silent, frameshift mutations almost invariably compromise protein function. Given the very deleterious nature of frameshift mutations, it is important to understand the mechanisms for generating insertions/deletions during DNA replication, as well as the editing functions that prevent fixation of such replication errors. This laboratory has developed a frameshift-specific assay system that allows a broad spectrum of frameshift events to be readily identified in the yeast Saccharomyces cerevisiae. This system is based on reversion of a defined +4 frameshift allele (lys2deltaBgl) at the LYS2 locus. Previous analyses of lys2deltaBgl reversion events in wild type, proofreading-defective (Exo-) and MMR-defective strains have demonstrated that almost all of the compensatory frameshifts are second site, 1 bp deletion events. This project extends the previous analyses and has three specific aims: 1. Previous analyses of lys2deltaBgl reversion events will be extended by constructing yeast strains that are defective in both of the pathways that remove replication errors: exonucleolytic proofreading and MMR. An examination of the lys2deltaBgl reversion spectrum in Exo-MMR- strains will allow a direct determination of the frameshift errors that are made during DNA replication. Comparison of the Exo-MMR-spectrum to the Exo-MMR+ spectrum will allow direct determination of the replication intermediates removed by the MMR machinery. Similarly, comparison of the Exo-MMR- spectrum to the Exo+MMR- spectrum will reveal replication errors subject to proofreading. 2. Frameshift spectra obtained in proofreading-defective (Exo-) mutants will be used to investigate the relative roles of yeast DNA polymerase II and DNA polymerase III in leading versus lagging strand synthesis during DNA replication. Reversion spectra obtained with lys2deltaBgl in its normal orientation on chromosome II will be compared with spectra obtained after inverting the locus, a process which switches the identities of the leading and lagging strands. If a given polymerase is specialized for either leading or lagging strand synthesis, then one would predict that the Exo- frameshift spectrum should change significantly upon reversal of the leading and lagging strands. In contrast, if a polymerase is involved in both leading and lagging strand synthesis, then the spectrum should not change significantly when the mutational target is inverted. 3. The lys2deltaBgl system allows examination of -1 frameshifts, but does not allow the detection of the other major class of frameshift events: 1 bp insertions. An alternative to the lys2deltaBgl system that will allow identification of predominantly + 1 frameshift events is being developed. This will be accomplished by subjecting the region of LYS2 containing the lys2deltaBgl reversion window to site-directed mutagenesis, which will convert the -1 frameshift window into a window for specifically identifying 1 bp insertions. As with the lys2deltaBgl -1 frameshift window, the molecular natures of +1 frameshifts in wild type, MMR-defective and proofreading-defective strains will be examined.
