Princeton University Library Catalog
- Vo, Linda [Browse]
- Senior thesis
- Zakian, Virginia [Browse]
- Princeton University. Department of Molecular Biology [Browse]
- Class year:
- 90 pages
- Summary note:
- Rrm3 is a 5’ to 3’ DNA helicase in Saccharomyces cerevisiae that has been implicated in resolving non-histone protein-DNA structures that are obstacles to replication fork progression. In the absence of Rrm3, replication fork pausing occurs at ~1400 sites in the genome, including inactive replication origins, rDNA, and tRNA genes. Prolonged pausing can result in genomic instability, fork collapse, or gross chromosomal rearrangements (GCRs), such as inversion, deletions, translocations, and amplifications. Although rrm3Δ cells have a ~30-100 fold increase in pausing at tRNA sites, rrm3Δ mutations are surprisingly not lethal. Therefore, we wanted to identify other proteins with redundant function to Rrm3 in resolving protein-DNA complexes. However, no efficient tool for screening complementary Rrm3 genes exists, and no Rrm3-backup gene has been identified to date. We developed a modified GCR assay as an efficient tool to screen proteins with complementary function to Rrm3 at tRNA genes, an rrm3Δ-sensitive site. Previously, no assay has been developed to measure the mutation rates at specific rrm3Δ-sensitive sites.
We screened four candidates for complementary Rrm3 function (Pif1, Rad5, Rad54, and Asf1) using the modified GCR assay. Although the candidate proteins screened did not show positive results, the construct has been shown to be effective in screening candidate proteins. This tool can be adapted for study of other candidate genes at tRNA genes or even other rrm3Δ-sensitive sites. Consistent with the literature on prolonged pausing at tRNA genes in rrm3Δ cells, we observed a small increase in GCR rate in the absence of Rrm3. We also observed a directionality-dependent increase in GCR rate for tRNA inserts, suggesting a transcription-dependent mechanism in replication fork progression. We propose a model where replication and transcription collision contribute to GCR events the rrm3Δ-sensitive tRNA gene.Since no in vitro studies can be conducted on Rrm3 due to an inability to purify the protein, identifying genes with complementary Rrm3 function can better inform our understanding of the mechanism of Rrm3 function.