A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair

Mol Cell Biol. 1995 Aug;15(8):4420-9. doi: 10.1128/mcb.15.8.4420.

Abstract

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Division / genetics
  • Cold Temperature
  • DNA Damage
  • DNA Mutational Analysis
  • DNA Polymerase II
  • DNA Polymerase III
  • DNA Repair*
  • DNA Replication* / drug effects
  • DNA-Binding Proteins
  • DNA-Directed DNA Polymerase / metabolism
  • Genes, Dominant
  • Homeodomain Proteins*
  • Minor Histocompatibility Antigens
  • Models, Molecular
  • Phenotype
  • Polyethylene Glycols / pharmacology
  • Proliferating Cell Nuclear Antigen / genetics*
  • Proliferating Cell Nuclear Antigen / immunology
  • Protein Binding
  • Protein Folding
  • Proto-Oncogene Proteins c-bcl-2*
  • Replication Protein C
  • Repressor Proteins*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / immunology
  • Saccharomyces cerevisiae Proteins*
  • Structure-Activity Relationship
  • Suppression, Genetic
  • Ultraviolet Rays / adverse effects

Substances

  • BCL2-related protein A1
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MATA1 protein, S cerevisiae
  • Minor Histocompatibility Antigens
  • Proliferating Cell Nuclear Antigen
  • Proto-Oncogene Proteins c-bcl-2
  • RFC1 protein, S cerevisiae
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Polyethylene Glycols
  • DNA Polymerase II
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase
  • Replication Protein C