In vivo analysis reveals that the interdomain region of the yeast proliferating cell nuclear antigen is important for DNA replication and DNA repair.
To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs-) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mmss mutations, the Cs- mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs- pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs- pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0651, USA.
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D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics; D003080:Cold Temperature; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D005656:Fungal Proteins / Q000235:genetics; D016195:G2 Phase; D015966:Gene Expression Regulation, Fungal; D005804:Genes, Lethal; D008938:Mitosis; D016296:Mutagenesis; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics*; D051818:Replication Protein C; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction
D000946:Antigens, Fungal; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D018809:Proliferating Cell Nuclear Antigen; C084903:RFC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D051818:Replication Protein C