Browsing by Subject "DNA Replication"

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    C-terminal flap endonuclease (rad27) mutations: lethal interactions with a DNA Ligase I mutation (cdc9-p) and suppression by proliferating cell nuclear antigen (POL30) in Saccharomyces cerevisiae.
    (2009-05) Karanja, Kenneth Kimani
    We recovered the flap endonuclease mutation rad27-K325* in a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its proliferating cell nuclear antigen (PCNA) interaction domain. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. Tests of mutation avoidance and DNA repair showed that rad27-K325* confers severe phenotypes similar to rad27Δ, rad27- A358* confers mild phenotypes and rad27-pX8 confers phenotypes intermediate between the other two alleles. rad27-K325* behaves similarly to rad27Δ in being lethal with exo1Δ and rad51Δ and not with rad2Δ. Interestingly, rad27-pX8 is lethal with rad51Δ, while rad27-A358* is lethal with rad51Δ at an elevated temperature. High copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Δ. rad27-K325* has an absolute lethality with the PCNA mutation pol30-90 that is not possessed by rad27Δ. These studies show the importance of the C-terminus of the flap endonuclease in mutation avoidance, and, by virtue of the initial screen, the role that PCNA plays in coordinating the entry of DNA ligase and the flap endonuclease in replication and repair.
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    Mcm10 regulates the turnover of DNA polymerase alpha.
    (2010-12) Haworth, Justin Christopher
    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.