All eukaryotic genomes contain simple repetitive DNA sequences. These repeat sequences are inherently instable. It is believed that this instability is caused by slippage of DNA polymerase through the repeat tract during replication. This rate of instability increases in yeast strains that are deficient in DNA mismatch repair. Significantly, tumor cells from certain forms of cancer are known to exhibit similar simple repeat length instability and have mutations in mismatch repair genes. Also, a number of heritable human neurological disorders are characterized by large expansions of triple repeat sequences. In order to help understand the mechanism underlying simple repeat instability, a study in the yeast Saccharomyces cerevisiae is proposed aimed at identifying components of the yeast DNA replication system that are important in maintaining simple repeat stability. Genes of the various yeast DNA polymerases and replication cofactors will be targeted for mutagenesis with primary emphasis on those regions corresponding to domains that are thought to affect polymerase processivity. A previously developed genetic assay that scores for the frequency of repeat tract alteration will be utilized to evaluate the effect that chosen mutants have on repeat instability. A selected sub-set of mutant DNA polymerases will be expressed, purified and assayed in vitro for activity and processivity.
