The proposed research is focused on understanding Saccharomyces cerevisiae DNA polymerase E's (pol E)role in DNA replication, regulating cell cycle checkpoints and repairing damaged DNA. The carboxyl terminal half of pol E is the non-catalytic domain that is necessary and sufficient for the survival of yeast. Mutations in the non-catalytic domain will be made and analyzed for defects in DNA replication, cell cycle checkpoints, and DNA repair. The mutant protein will be purified and analyzed for polymerase activity, forming the holoenzyme complex, and binding single-stranded DNA. The non-catalytic domain will also be tested for association with Trf4, Trf5, or Trf4/Trf5- proteins linked to sister chromatid cohesion, recombination, or repair. PCNA will be tested for its ability to recruit pol E to repair damaged DNA. The hypothesis that the non-catalytic domain is important for interacting with other proteins and single-stranding DNA will be tested and applied to understand the biological function of pol E.
| Dua, Rajiv; Levy, Daniel L; Li, Caroline M et al. (2002) In vivo reconstitution of Saccharomyces cerevisiae DNA polymerase epsilon in insect cells. Purification and characterization. J Biol Chem 277:7889-96 |
