Transcription has a strong stimulatory effect on mutagenesis in budding yeast, and most transcription-associated mutations are short deletions that remove one copy of a 2-5 bp tandem repeat. These signature deletions require the activity of Top1, a topoisomerase that removes transcription-associated supercoils by nicking one strand of DNA, passing the intact strand through the nick, and then resealing the nick. A similar, short-deletion signature has been associated with the failure to remove ribonucleotides (rNMPs) misincorporated into DNA, and this signature likewise depends on Top1 activity. Though there is overlap between where transcription-dependent and rNMP-dependent deletions accumulate, the respective hotspots are not entirely coincident. We thus propose that there are two distinct types of Top1-generated lesions that lead to deletions: (1) a Top1 cleavage complex (Top1cc) that becomes trapped on DNA during the normal cleavage-ligation cycle and (2) an irreversible nick created when Top1 cleaves at an rNMP. Both types of lesions, however, are proposed to be processed into a small gap that resides within the relevant tandem repeat. Subsequent misalignment between repeats on the complementary strands will bring the ends together to facilitate ligation. The proposed studies will characterize the mechanisms of Top1-dependent mutagenesis at each of the two types of deletion hotspots.
In Aim 1, we will map Top1 cleavage sites in vitro, and then use this information to define the positional relationship between the cleavage site and the tandem repeat where deletions occur in vivo.
Aim 2 will use a candidate gene approach to identify proteins that process Top1-generated lesions into a small gap.
Aim 3 will focus on rNMP- dependent hotspots;the DNA polymerase that inserts rNMPs will be identified and the relevance of cell-cycle phase to mutagenesis will be examined. Finally, Aim 4 will define factors that trap the Top1cc during transcription and will explore the mutagenic potential of camptothecin, a chemotherapeutic drug that stabilizes the Top1cc. Given the universality of DNA structure and basic DNA metabolic processes, results in the yeast system will be relevant to issues of genome stability in higher eukaryotes.
Mutations provide the raw material for evolutionary processes and are causative in a number of human diseases, especially cancer. The proposed experiments will use budding yeast as a model system to explore mutagenesis that derives specifically from the activity of Top1, a topoisomerase that resolves the supercoils associated with transcription. These studies will explore the general mechanism of Top1-dependent mutagenesis as well as the mutagenic potential of camptothecin, a chemotherapeutic drug that specifically targets Top1.
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