DNA repair and surveillance mechanisms are essential for the maintenance of eukaryotic genome integrity. In humans, these mechanisms protect somatic tissue from the accumulation of the genetic changes associated with cancer and ensure that the informational content is stably transmitted from one generation to the next. My laboratory uses the yeast Saccharomyces cerevisiae as a model genetic system to examine the regulation of mitotic/somatic eukaryotic genome stability. Current studies focus on (1) understanding mechanisms of spontaneous and induced mutagenesis (2) understanding the regulation of recombination fidelity, and (3) understanding the relationship between transcription and genetic instability.

Mechanisms of mutagenesis
Mutations originate either as mistakes made by a replicative DNA polymerase when copying an undamaged DNA template, or during cellular attempts to deal with DNA damage. In yeast, most damage-associated mutagenesis is due to activity of the error-prone translesion DNA polymerase Polζ. While Polζ is dispensable in yeast, it is essential in mammalian cells, probably allowing the bypass of lesions encountered during DNA replication. Not only is it critical for Polζ to be efficiently recruited when needed for lesion bypass, but it also is important to limit the access of this error-prone polymerase to undamaged DNA templates. Using a frameshift-specific reversion assay, we have discovered a distinct mutational signature associated with the activity of yeast Polζ, thereby providing us with a unique tool to study the regulation of translesion synthesis (TLS) with regard to spontaneous as well as induced DNA damage.

Our current studies are focused on understanding how Polζ is recruited to bypass DAN lesions, and whether the mechanism of lesion bypass is the same during leading versus lagging strand synthesis. In addition to using frameshift-specific reversion assays to study Polζ activity, we are also using a suppressor tRNA-based (SUP4-o) forward mutation assay to examine the bypass of lesions by the TLS polymerase Polη. The SUP4-o system detects any genetic change that inactivates the suppressor tRNA, and is particularly useful for looking at base substitutions in a relatively unbiased manner. As in our studies with Polζ, we are focusing on genetically defining the regulation of Polη-dependent lesion bypass.

Regulation of recombination fidelity
When mitotic recombination occurs, it is important that it involve either identical sequences on sister chromatids or homologous chromosomes, as recombination between dispersed repeated sequences can lead to a variety of detrimental genome rearrangements. To study the relationship between sequence divergence and homologous recombination, we have developed an intron-based recombination assay that allows the degree of identity between interacting sequences to be varied over a very broad range. Using this system, we have demonstrated that the yeast mismatch repair (MMR) system is exquisitely sensitive to the presence of mismatches in recombination intermediates; a single potential mismatch is sufficient to reduce recombination in an MMR-dependent manner. DNA sequence analysis of recombination products is consistent with a model in which the MMR machinery exerts its anti-recombination activity by blocking the extension of mismatch-containing heteroduplex DNA. Our current efforts are focused on understanding the molecular mechanism whereby the MMR system blocks recombination, and on determining whether sequence divergence influences the resolution of recombination intermediates as crossover versus noncrossover products.

Transcription and genome stability
Because the DNA metabolic processes of transcription, replication and repair are not temporally separated, one process has the potential to influence the occurrence of another. We have found that the stability of DNA is related not only to its primary sequence, but also is influenced by its level of transcription. Using the highly inducible pGAL or pTet system in yeast, we have documented that high levels or transcription are associated with elevated mutation and recombination rates. In the case of transcription-associated mutagenesis, the level of mutagenesis is directly proportional to the level of transcription, and direction of replication fork movement relative to that of RNA polymerase affects mutagenesis at the molecular level. Current efforts focus on understanding the molecular mechanisms responsible for transcription-associated genome instability.