Characterization of DNA ligase from the fungus Coprinus cinereus.
DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP-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(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively. This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n 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 mass, immuno-reactivity, K(m) 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.
Matsuda
S
S
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Japan.
Sakaguchi
K
K
Tsukada
K
K
Teraoka
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002384:Catalysis; D003302:Coprinus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D011088:DNA Ligases