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International audience; 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.

International audience; The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.

International audience; 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.