THE B-SUBUNIT OF THE DNA-POLYMERASE ALPHA-PRIMASE COMPLEX IN SACCHAROMYCES-CEREVISIAE EXECUTES AN ESSENTIAL FUNCTION AT THE INITIAL-STAGE OF DNA-REPLICATION

The four-subunit DNA polymerase cy-primase complex is unique in its ab ility to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA repli cation, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the comple x is unknown, but the Saccharomyces cerevisiae POL12 gene, which encod es this protein, is essential for cell viability. We have produced dif ferent pol12 alleles by in vitro mutagenesis of the cloned gene. The i n vivo analysis of our 18 pol12 alleles indicates that the conserved c arboxy-terminal two-thirds of the protein contains regions that are es sential for cell viability, while the more divergent NH2-terminal port ion is partially dispensable. The characterization of the temperature- sensitive pol12-T9 mutant allele demonstrates that the B subunit is re quired for in vivo DNA synthesis and correct progression through S pha se. Moreover, reciprocal shift experiments indicate that the POL12 gen e product plays an essential role at the early stage of chromosomal DN A replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.