A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

Abstract

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 Xgtll 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 PRI2. 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 PRJ2 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.A DNA polymerase-primase complex with a highly conserved polypeptide structure has been found in a number of eucaryotic organisms phylogenetically as distant as yeasts and humans. A complete understanding of the functions of such a complex has not yet been achieved, but several data suggest that it plays a crucial role not only in priming and elongation of the Okazaki fragments but also in the initiation step of DNA replication at an origin (reviewed in references 4 and 15). DNA polymerase activity is associated with the highest-molecular-size polypeptide of the complex (170 to 180 kilodaltons [kDa]), whereas the two smallest polypeptides (-60 and -50 kDa, respectively) are correlated with DNA primase activity. A fourth polypeptide, of 70 to 80 kDa, whose function is still unknown, is also present in the complex (4, 15).To better understand the functions and interactions in this protein complex essential for DNA replication, we undertook the cloning and characterization of the genes encoding the different protein species in Saccharomnvces cerevisiae. The genes coding for DNA polymerase I, POLI. (13. 19,26), and the small primase polypeptide, PRII (20,30) essential for cell viability and is directly related to DNA primase activity (20). Recently, we used a highly specific affinity-labeling procedure to map the catalytic site of yeast DNA primase and found that both p48 and p58 participate in formation of the primase active center, although the ATPbinding site is exclusively located on the p48 subunit (Sa).In this paper, we report the isolation and characterization of the yeast PRI2 gene, encoding the p58 polypeptide. We show that the gene is unique in the haploid yeast genome and is transcribed in a 1.7-kb mRNA that appears to fluctuate in amount during the cell cycle. The gene encodes a protein with a calculated molecular size of 62,262 Da that is essential for cell viability and is directly related to DNA primase activity, thus confirming previous biochemical findings. Fig. 6). Plasmid pMF18 is a ...

A group of interacting yeast DNA replication genes.

“…These genes have the small 6-bp sequence, "ACGCGT" upstream of their promotors. Most of the genes in this group are thought to be involved with DNA synthesis or its regulation; these include POLl (DNA ot-polymerase), cdc2 (DNA ~-polymerase), cdc9 (DNA ligase), SSB1 (single-stranded binding protein), CDC7 (protein kinase), CDC8 (thymidylate kinase), and PRI1 and PRI2 (subunits of the DNA ot-polymerase) (Johnston et al 1987;Foiani et al 1989). Although it is unlikely that this sequence is solely responsible and sufficient for cellcycle transcriptional regulation, it may play a role in conjunction with other regulatory factors.…”

DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase

“…Degenerate primers derived from bovine peptide sequence (p68 4k and p68 5k) from conserved p68 amino acid regions (F1 and F2) and from cDNAs (F3k, F4k, F5 and F6). (C) Comparison of yeast, mouse and human p58 subunits (Foiani et al, 1989a). Degenerate primers derived from conserved regions, p58a and p58bk; non-degenerate primers derived from obtained cDNAs, p58c and p58dk.…”

“…The cDNAs encoding the subunits of DNA-polymerasea-primase from several eukaryotes have been cloned and sequenced; cDNAs for all four subunits from yeast and mouse, p180 DNA polymerase a and p73 subunit from Drosophila, human p180 DNA polymerase a and, recently, p68 (Lucchini et al, 1987;Pizzagalli et al, 1988;Wong et al, 1988;Foiani et al, 1989a;Prussak et al, 1989a;Brooke et al, 1991;Damagnez et al, 1991;Hirose et al, 1991;Cotterill et al, 1992;Melov et al, 1992;Collins et al, 1993;Miyazawa et al, 1993). Sequence comparisons showed that p180 DNA polymerase a is distantly related to DNA polymerases from bacteriophages, viruses and bacteria and contains several regions that are highly conserved among the mammalian, Drosophila and yeast p180 subunits (Braithwaite and Ito, 1993).…”

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 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).…”