Haworth, Justin Christopher2011-02-082011-02-082010-12https://hdl.handle.net/11299/99897University of Minnesota Ph.D. dissertation. December 2010. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Anja-Katrin Bielinsky. 1 computer file (PDF); ix, 165 pages. Ill. (some col.)Accurate and efficient duplication of the genome exactly once per cell cycle is crucial for prolonged health of an organism. Minichromosome maintenance (Mcm) 10 is an essential, highly conserved replication factor with multiple functions at the fork. Critical for Mcm10's role at the fork is its DNA binding activity. Utilizing the crystal structure of the conserved internal domain of X. laevis (x) Mcm10, we identified residues that, when mutated, significantly reduced xMcm10's ability to bind DNA in vitro. Importantly, the corresponding mutations in S. cerevisiae (sc) Mcm10 resulted in reduced viability after exposure to hydroxyurea, a drug that causes replication fork stalling. This suggests that the DNA binding activity of Mcm10 is important in vivo for fork stabilization during replication stress. In addition to its DNA binding activity, work from our laboratory has shown that Mcm10 regulates the stability of DNA polymerase (pol) alpha/primase, the only enzyme capable of de novo DNA synthesis, in yeast and humans. In the absence of Mcm10, Cdc17, the catalytic subunit of pol alpha, is rapidly degraded. We have determined that Cdc17 degradation is dependent on the proteasome via Ubc4 and Not4 and disrupting this degradation pathway results in elevated steady-state levels of Cdc17. Furthermore, Cdc17 shows synthetic dosage lethality with not4 cells. Importantly, overexpression of Cdc17 and Mcm10 causes slow growth, an increased mutation rate, and microsatellite-mediated gross DNA rearrangements, suggesting that regulated turnover of Cdc17 is crucial to maintain genome stability.en-USCell CycleDNA ReplicationProteasomal DegradationBiochemistry, Molecular Bio, and BiophysicsThesis or Dissertation