Regulation of mitotic genome stability in yeast.

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A low level of genetic instability is required for adaptation and evolution, but such instability is also a potent driver of human disease. Research in my lab focuses on genetic identification and molecular characterization of processes that contribute to mitotic genome instability as well as DNA repair processes that promote genome stability. The proposed research will primarily use budding yeast (Saccharomyces cerevisiae) as a model to explore the repair of DNA strand breaks and how this impacts genome integrity. Double-strand breaks (DSBs) are among the most detrimental of DNA lesions and are repaired either by homologous recombination (HR), which uses an intact duplex as a repair template, or by nonhomologous end joining (NHEJ), which directly rejoins broken ends. Although both are inherently high-fidelity processes, HR can result in loss of heterozygosity or can engage dispersed repeated sequences to generate genome rearrangements. In the case of NHEJ, end processing prior to ligation produces small-scale changes at the junction while joining the ends of different DSBs generates genome rearrangements. Endonuclease-generated DSBs that have different end polarities will be used to initiate HR between sequence-diverged substrates on different chromosomes. Comparative analyses of HR product types and their strand compositions will reveal how end structure affects mitotic HR intermediates and mechansims. The effects of large sequence discontinuities at the site of an initiating DSB will be examined. In addition to use of sequence-specific enzymes to create targeted DSBs, topoisomerases break and rejoin DNA strands to resolve topological problems that arise during transcription and replication. These enzymes form a covalent link with one end of a nick; stabilization of cleavage intermediates with chemotherapeutic drugs leads to persistent breaks that are highly toxic. We previously described a short-deletion signature of Top1 (a type I enzyme that nicks one DNA strand) and defined the associated molecular mechanism. We recently discovered that Top2 (a type II enzyme that nicks both strands to create a DSB) initiates the formation of de novo duplications through the NHEJ pathway. We will examine how the mechanism of protein removal from DNA ends and how the presence of ribonucleotides embedded in DNA affect Top2-dependent mutagenesis. Similar duplications are found in tumor cells with a mutant form of TOP2a, and this mutant protein will be modeled in yeast. Building on our long-term interests in recombination and mutagenesis in the budding yeast experimental system, we recently expanded studies to include mutagenesis in the human fungal pathogen Cryptococcus deneoformans. Cryptococcus must rapidly adapt to hostile conditions when it transitions from the environment to the human host, and heat tolerance is critical for pathogenesis. Using a forward mutation assay, we found that a temperature shift mimicking the environment-human transition is associated with the mobilization of transposable elements (TEs). Futures studies will focus on a more global analysis of temperature-dependent TE movement and the molecular mechanism(s) of mobilization.