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.

Karanja, Kenneth Kimani
2009-05
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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.

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2009-05

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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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University of Minnesota Ph.D. dissertation. May 2009 Major: Biochemistry, Molecular Biology, and Biophysics. Advisor: Dennis Livingston. 1 computer file (PDF); vi, 141 pages.

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Karanja, Kenneth Kimani. (2009). 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.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/52143.

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