Yeast DNA Primase Genes Affect Different Aspects of DNA Metabolism and Interactions in the DNA Polymerase a-Primase Complex

Different pril and pri2 conditional mutants of Saccharomyces cereuisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and errorprone repair, due to defective DNA replication in the primase mutants. Furthermore, pril and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri l and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase a, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase a-primase complex. I N the yeast Saccharomyces cerevisiae, as in all eukaryotic systems analyzed so far, a DNA primase activity is associated with DNA polymerase a (pola) in a four-subunit complex that appears to play a major role in initiation of DNA replication at an origin and in priming and elongation of Okazaki fragments on the lagging strand (LEHMAN and KAGUNI 1989; TSURIMOTO, MELENDY and STILLMAN 1990). The yeast complex can be separated into two fractions, one of which, containing the p48 and p58 polypeptides, is able to synthesize short RNA primers (9-1 1 nucleotides in length) that can be elongated by pola (PLEVANI et al. 1985; BROOKS and DUMAS 1989). Cloning and characterization of the PRIl and PRI2 genes encoding, respectively, the p48 and p58 protein species, have shown that they are both unique in the haploid yeast genome and essential for cell viability, and that the amino acid sequence of their encoded products is highly homologous to that of the corresponding mouse primase polypeptides (PLEVANI, FRANCESCONI and LUCCHINI 1987; FOIANI et al. 198913; PRUSSAK, ALMAZAN and TSENC 1989; FRANCESCONI et al. 199 1). Earlier studies with anti-p48 and anti-p58 antibodies and an affinity labeling procedure suggest that both proteins are necessary for optimal DNA primase activity (FOIANI et al. 1989a). However, the inability to purify each primase subunit in an isolated form in any eukaryotic system has until now limited the possibility Genetics 133 183-1 91 (February, 1993) to use in vitro reconstitution studies to further analyze their specific role in determining enzyme activity and their interactions. To address these questions and to gain some information about the function of DNA primase in different aspects of DNA metabolism, we have produced conditional pril and pri2 mutants by random mutagenesis of the cloned genes and replacement of the wild-type copy at the corresponding chromosomal locus (FRANCESCONI et al. 199 1). Preliminary characterization of different recessive alleles, i . e . , the leaky and temperature-sensitive (ts) pril-1, the cold-sensitive (cs) pril-2, the ts pri2-1 and the ts pri2-2 alleles, which all carry single base-pair substitutions causing a change in amino acid residues conserved in the corresponding mouse polypeptides, has allowed us to assess the essential role of DNA primase in DNA replication in vivo. In fact, the primary defect in all these mutants, after shifting to n npermissive temperature, is an impairment in DNA synthesis, ranging between a complete arrest in the pri2-1 mutant to only a doubling of the time needed to complete one round of DNA replication in the prz2-2 mutant. These defects are correlated with cell growth arrest or reduced growth rate, accumulation of dumbbell-shaped cells and inability to synthesize appropriate amounts of high molecular weight DNA molecules (FRANCESCONI et al. 1991).