The four-subunit DNA polymerase a-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We Cloning of the S. cerevisiae POLl, PRII, and PR12 genes, which encode the p180, p48, and p58 subunits of the yeast complex, respectively, and the study ofpoll, pril, and pn2 lethal and conditional alleles have been essential in establishing the roles of the corresponding gene products in mitotic DNA replication and in identifying functional domains in the polymerase and primase polypeptides (8,26,29,42,51,54). Moreover, poll, pnil, and pn2 conditional mutants are defective in premeiotic DNA synthesis and show an enhanced rate of intrachromosomal recombination and spontaneous mutation, which is generally correlated with the severity of their defects in cell growth and DNA synthesis (9,41,42). Since these mutant strains fail to accumulate high-molecular-weight DNA products (9, 29), the formation of nicked and gapped replicated DNA molecules might be responsible for the hyperrecombination phenotype usually found in yeast DNA synthesis mutants (33).No enzymatic activity has been found to be associated with the 86-kDa protein species (B subunit), which is tightly bound to the p180 polypeptide, and the physiological role of p86 is presently unknown (7). In vitro reconstitution studies with purified components indicate that p86 is not required for polymerase-primase interaction, and its presence does not change the catalytic properties of Pol a (6, 7). Recently, it has been proposed that the human p86 homolog might serve as a molecular tether during DNA replication, because this subunit mediates the in vitro interaction between the human Pol a-primase complex and T antigen bound to the SV40 origin of replication (14).As a first step in identifying the physiological role of the B subunit of the yeast Pol a-primase complex in vivo, we have mutagenized by the two-codon insertion mutagenesis method (3) the essential POL12 gene, which was recently found to encode the p86 polypeptide (7, 36a). The analysis of 923

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The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA 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.

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