Characterization of DNA Ligase from the Fungus Coprinus cinereus

Abstract

) -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...

Kinetic Mechanism of the Mg2+-dependent Nucleotidyl Transfer Catalyzed by T4 DNA and RNA Ligases

“…The typical intracellular concentration of ATP is around 1-3 mM (32), indicating that the main fraction of nucleotide is present in the mono-magnesium-coordinated form. From the values shown in Table III, the intracellular concentration of the di-magnesium-coordinated ATP can be estimated as 60 -170 M, which is either higher or in the order of the K m of many ATP-dependent enzymes, including T4 DNA and RNA ligases (20,(33)(34)(35)(36)(37)(38)(39). It has been shown that the monomagnesium-coordinated form of ATP exists in aqueous solution as a mixture of rapidly exchanging mono-, bi-, and tridentate structures, with ␤-and ␥-phosphoryls being the main ligands (40,41).…”

Life History and Developmental Processes in the BasidiomyceteCoprinus cinereus

“…Chromosome translocations are common in C. cinereus (176,301,392,541). Reciprocal translocations have been mapped between chromosomes 3 and 5 and between chromosomes 1 and 9 (176,392).…”

“…The deletions led to an in-frame fusion of the HD2 gene a2 from the a gene pair with variable parts of the HD1 gene d1 from the d gene pair. In consequence, covalently linked proteins are formed whose components normally do not interact with each other (12,13,259,301,378). Interestingly, both former N-terminal domains become superfluous in such HD1-HD2 fusion proteins, consistent with their function in recognition and discrimination (13,259).…”

DNA ligase IV from a basidiomycete, Coprinus cinereus, and its expression during meiosis

“…Assays of DNA ligase activity were performed as described previously (Matsuda et al, 1996;Teraoka et al, 1993) with minor modifications. Adenylation was performed in a 20 ml reaction mixture containing 60 mM Tris/HCl (pH 7?8), 10 mM MgCl 2 , 5 mM DTT, 50 mg BSA ml 21 and 1 mCi [a-32 P]ATP (3000 Ci mmol 21 ; 110 TBq mmol 21 ) and 2 ml of the ligase fraction.…”

“…NaPP i treatment was performed in the presence of 0?5 mM NaPP i in the reaction buffer. DSB ligation reactions were performed using a linearized pUC119 DNA with BamHI or SmaI as described previously (Matsuda et al, 1996).…”