Background: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein.
A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)12-18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.
) -EJB 95 2128/4 DNA ligase was highly purified from the fungus Coprinus cinereus at the meiotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [-"PIAMP-labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures. K, values for ATP and (dT),, hybridized with (dA),, were 1.5 pM and 90 nM, respectively. This enzyme was capable of joining (dT),,] . (rA),3 and (rA),2-ls .(dT), as well as (dT),, . (dA),, and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular macs, immuno-reactivity, K, value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C.cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.Keywords: DNA ligase; Coprinus cinereus ; meiosis ; mycelium.DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single-strand or double-strand breaks of duplex DNA. It is therefore an essential element of the coordinated multienzyme processes of DNA replication, DNA excision repair, DNA doublestrand break repair and DNA recombination. Genetic studies in Escherichia coli and yeasts have demonstrated that a single species of DNA ligase is essential for DNA replication, repair and recombination [l -41. In contrast, mammalian and Drosophilu melanogaster embryo cells contain at least two different species of the enzyme [5-131. DNA ligase I activity and the gene expression are higher in rapidly dividing cells than in nonproliferating cells, implying a role for DNA ligase I in DNA replication [14-181. In fact, complementation of the conditional lethal phenotype of Succharonzyces cerevisiue cdc9 DNA ligase mutant by human DNA ligase I cDNA demonstrates that this enzyme is the key enzyme for joining Okazaki fragments during laggingstrand DNA synthesis [19]. Direct evidence has been obtained that the human cell line 46BR, which has two missense mutations in different alleles of the DNA ligase I gene, is defective not only in DNA replication but also in DNA excision repair In contrast to the animal kingdom, much less is known about DNA ligase in fungi and plants, though earlier reports showed the presence of ATP-dependent DNA...
A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.
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