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The monoclonal antibody (mAb) 21A6, which specifically inhibits yeast DNA primase activity, has been used to verify whether only one of the two polypeptides of heterodimeric DNA primase (48 and 58 kDa) was responsible for DNA primase function in vitro. Immunoaffinity chromatography of a crude extract from cells of Saccharomyces cerevisiae on a mAb 21A6 protein A-Sepharose 6B column allowed the purification of the p48 primase polypeptide in an isolated form. This polypeptide was not derived through the dissociation of the four-subunit DNA polymerase alpha-primase complex, which can be purified from the same extract by affinity chromatography with a mAb recognizing the DNA polymerase alpha polypeptide. Therefore, free p48 was already present in the yeast extract and, possibly, within the cell. Isolated p48, devoid of any detectable p58 subunit, was sufficient for RNA primer synthesis, although free primase appeared to extend RNA primer-monomers to primer-multimers less efficiently. Primase activity associated with free p48 was highly unstable, indicating that although p48 bears the catalytic site, its association with the other polypeptides of the polymerase-primase complex plays an important role in stabilizing enzyme activity.

Eukaryotic DNA primases are composed of two distinct subunits of 48-50 and 58-60 kDa. The amino acid sequences derived from the nucleotide sequences of the cloned genes are known only for the yeast and mouse polypeptides, and the extensive homology between the corresponding mouse and yeast subunits suggests conservation of functional domains. We were able to express in Saccharomyces cerevisiae the homologous and mouse primase-encoding genes under the control of both the constitutive ADH1 and the inducible GAL1 strong promoters, thus obtaining strains producing relevant amounts of the different polypeptides. In vivo complementation studies showed that neither one of the wild-type mouse primase-encoding genes was able to rescue the lethal or temperature-sensitive phenotype caused by mutations in the yeast PRI1 or PRI2 genes, indicating that these proteins, even if structurally and functionally very similar, might be involved in critical species-specific interactions during DNA replication.

To assess the role of eukaryotic DNA primase in vivo, we have produced conditional and lethal point mutations by random in vitro mutagenesis of the PR11 and PR12 genes, which encode the small and large subunits of yeast DNA primase. We replaced the wild-type copies of PRI1 and PRI2 with two pri1 and two pri2 conditional alleles. When shifted to the restrictive temperature, these strains showed altered DNA synthesis and reduced ability to synthesize high molecular weight DNA products, thus providing in vivo evidence for the essential role of DNA primase in eukaryotic DNA replication. Furthermore, mapping of the mutations at the nucleotide level has shown that the two pri1 and two pri2 conditional alleles and one pri2 lethal allele have suffered single base-pair substitutions causing a change in amino acid residues conserved in the corresponding mouse polypeptide.

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.