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International audience; The DNA polymerase alpha enzymes from human, and budding (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are homologous proteins involved in initiation and replication of chromosomal DNA. Sequence comparison of human DNA polymerase alpha with that of S. cerevisiae and S. pombe shows overall levels of amino acid sequence identity of 32% and 34%, respectively. We report here that, despite the sequence conservation among these three enzymes, functionally active human DNA polymerase alpha fails to rescue several different conditional lethal alleles of the budding yeast POL1 gene at nonpermissive temperature. Furthermore, human DNA polymerase alpha cannot complement a null allele of budding yeast POL1 either in germinating spores or in vegetatively growing cells. In fission yeast, functionally active human DNA polymerase alpha is also unable to complement the disrupted pol alpha::ura4+ allele in germinating spores. Thus, in vivo, DNA polymerase alpha has stringent species specificity for initiation and replication of chromosomal DNA.

International audience; DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.

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; Peplomycin, a bleomycin-related cytostatic agent, was tested on DNA polymerases and DNA ligase. These enzymes were purified from normal human immunocompetent cells (thymocytes and lymphocytes) and from peripheral blast cells from different kinds of acute lymphoblastic and acute non-lymphoblastic leukemia. At low concentration ranges (1-25 microM) this compound was found to strongly inhibit polymerase alpha and ligase from leukemic cells while being less effective on the enzyme activity from normal thymocytes and lymphocytes. At the DNA level, low concentrations of peplomycin resulted in the induction of dose-dependent single-stranded breaks. The incubation of peplomycin (5 microM) with plasmid DNA resulted in its degradation as observed by agarose gel electrophoresis. Lowering the peplomycin concentration showed that ligase inhibition takes place prior to this phenomenon. The decreased formation of the ligase--adenylate complex under the effect of peplomycin is consistent with a direct interaction between the drug and the enzyme. These results are discussed in terms of possible selective cytostatic effects of peplomycin on leukemic cells.