Schizosaccharomyces pombe replication and repair proteins: Proliferating cell nuclear antigen (PCNA)

Schizosaccharomyces pombe has a cell cycle progression with distinctive pha ses that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear an tigen (PCNA) of S. pombe to demonstrate how the function of this protein in both DNA replication and repair can be assessed by genetic and biochemical approaches. We de scribe a method of introducing site-specific mutations i nto the fission yeast PCNA gene pcn1(+). The in vivo effects of these pcn1 mutants in a strain with a null pcn1 background are described and their in vitro biochemical properties are characterized. Mutants described here are those that are defective in enhancing processivity of DNA polymerase delta, show temperature-sensitive growth, and have increased sensitivity to hydro xyurea (HU), UV and gamma irradiation, and methyl methanesulfonate (MMS). T hree mutants that show reduced growth rate in vivo and decreased capacity t o enhance polymerase delta DNA synthetic activity and processivity in vitro -pcn1-1, pcn1-5, and pcn1-26-are described as examples of using a genetic a pproach to identify the biochemical function of replication proteins. One c old-sensitive growth allele, pcn1-3, that has a recessive cold-sensitive cd c phenotype and shows sensitivity to HU and UV and gamma irradiation is use d as an example of using the genetic approach to reveal the function of rep lication proteins in repair. The power of combining both biochemical and ge netic disciplines is emphasized. Methods for site-directed mutagenesis, in vitro analysis of mutant proteins, and in vivo characterization of mutants in response to UV or gamma irradiation, MMS, HU, and temperature, as well a s genetic epistasis are de scribed. Locations of functionally significant r esidues on the PCNA tertiary structure are summarized. (C) 1999 Academic Pr ess.