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 (PCN A) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs-) and 31 methyl methanesulfonate-sen sitive (Mms(s)) mutations of the PCNA gene (POL30) in Saccharomyces ce revisiae. Unlike the Mms(s) mutations, the Cs- mutations are strikingl y clustered in the interdomain region of the three-dimensional PCNA mo nomer structure. At the restrictive temperature, the Cs- pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA c ontent. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through 3 phase at the restrictive temperature. DNA repair defects are revealed by the observ ation that Cs- pol30 mutants are very sensitive to the alkylating agen t MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosoma l DNA in pol30 cells by velocity sedimentation gradients shows that po l30 cells accumulate single-stranded DNA breaks at the restrictive tem perature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.