Schizosaccharomyces pombe Replication and Repair Proteins: Proliferating Cell Nuclear Antigen (PCNA)

• Published in: Methods (San Diego, Calif.) Vol. 18; no. 3; pp. 335 - 348
• Main Authors: Arroyo, Martha P., Wang, Teresa S.-F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.1999
• Subjects: Cell Cycle; Cell Division; DNA Polymerase III - genetics; DNA Repair - genetics; DNA Replication - genetics; Fungal Proteins - genetics; Hydroxyurea - pharmacology; Methyl Methanesulfonate - pharmacology; Models, Molecular; Mutagenesis, Site-Directed; Mutagens - pharmacology; Phenotype; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - genetics; Proliferating Cell Nuclear Antigen - radiation effects; Protein Structure, Tertiary; Schizosaccharomyces - genetics; Temperature
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1006/meth.1999.0795

Schizosaccharomyces pombe has a cell cycle progression with distinctive phases that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear antigen (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 describe a method of introducing site-specific mutations into 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 δ, show temperature-sensitive growth, and have increased sensitivity to hydroxyurea (HU), UV and γ irradiation, and methyl methanesulfonate (MMS). Three mutants that show reduced growth rate in vivo and decreased capacity to enhance polymerase δ DNA synthetic activity and processivity in vitro—pcn1-1, pcn1-5, and pcn1-26—are described as examples of using a genetic approach to identify the biochemical function of replication proteins. One cold-sensitive growth allele, pcn1-3, that has a recessive cold-sensitive cdc phenotype and shows sensitivity to HU and UV and γ irradiation is used as an example of using the genetic approach to reveal the function of replication proteins in repair. The power of combining both biochemical and genetic 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 γ irradiation, MMS, HU, and temperature, as well as genetic epistasis are described. Locations of functionally significant residues on the PCNA tertiary structure are summarized.