Brewer, Bonita JMartin, Rebecca2021-10-292021-10-292021Martin_washington_0250E_23534.pdfhttp://hdl.handle.net/1773/48075Thesis (Ph.D.)--University of Washington, 2021DNA replication is an essential process that is highly conserved in form and function throughout eukaryotes. However, budding yeast differ from higher eukaryotes in that their origins of replication are defined by sequence. This unique feature has long been a tool in the study of replication, but what drives origin sequence-specificity remained unclear until cryo-EM structures of the Saccharomyces cerevisiae Origin Recognition Complex (ORC) revealed base-specific contacts between origin DNA and ORC subunits 1, 2, and 4. Orc4 makes the most substantial contact through a long unstructured loop capped by an alpha helix termed the insertion helix (IH). I tested the hypothesis that the Orc4-IH drives sequence specificity of budding yeast origins by replacing the Orc4-IH in S. cerevisiae with the corresponding sequence in Lachancea waltii and by deleting the alpha helix of the Orc4-IH. I found that the chimeric Orc4-IH enabled recognition of L. waltii specific plasmids while the Orc4-IH deletion reduced plasmid retention. Both strains exhibited altered genome-wide origin usage and pre-replication complex loading. In addition, I made a complementary chimeric mutation in Orc1 and tested its impact on origin sequence specificity alone and in combination with the chimeric Orc4-IH. I found that on its own, the Orc1 chimera is unable to recognize L. waltii plasmids but does show altered origin usage. However, when in conjunction with the Orc4-IH chimera, L. waltii plasmid retention was even greater, as was S. cerevisiae plasmid retention and origin function. It appears that while Orc4 alone is sufficient to alter origin specificity, complementary changes to Orc1 may stabilize overall ORC binding. In addition to this work, I also helped obtain evidence for intermediates of the origin dependent inverted repeat amplification (ODIRA) model in a yeast strain engineered to select for interchromosomal amplification events. We found that genetic alterations consistent with ODIRA did occur and that induction of double stranded breaks did not increase these events, further suggesting that these events were caused by a replication error rather than through repair of double-stranded DNA breaks.application/pdfen-USCC BY-NC-SADNA replicationODIRAORCorigin licensingS. cerevisiaeMolecular biologyGeneticsMolecular and cellular biologyThesis