Purification and characterization of DNA ligase II from Drosophila melanogaster

Abstract

Drosophila melanogaster contains DNA ligases I and 11. The activity of DNA ligase I is especially high during early embryonic periods, but decreases rapidly afterwards. Although the activity of DNA ligase I1 is low, it persists throughout all developmental stages. The specific activity of DNA ligase I1 is high in embryos, but the total activity per body mass was highest in pupae. To characterize the properties of DNA ligase I1 further and to clarify its differences from DNA ligase I, DNA ligase I1 was prepared from pupae of D . melanogaster. The enzyme was purified about 3200-fold by ammonium sulfate fractionation (40 -70% saturation), phosphocellulose (P1 1) and Ultrogel column chromatography. Some of the properties have been reported previously. The isoelectric point of DNA ligase I1 was 6.4 while those of DNA ligase I were 4.9 and 5.8. The optimum pH of DNA ligase I1 was 7.8-8.1 but 8.0-8.5 for DNA ligase I.The molecular masses of DNA ligase I1 adducts with AMP were determined as 90 and 70 kDa. These adducts were degraded to 42 and 14.4 kDa by trypsin digestion. For preparation of monoclonal antibodies, a mouse was immunized with the purified enzyme. Two clones, 10-6 and 3-3 IgM, were obtained and purified from mouse ascites.These antibodies showed both binding and neutralizing activities toward DNA ligase I1 from D. melanoguster, but did not react with DNA ligase I from the same origin.These results showed clearly that DNA ligases I and I1 have different properties and suggest they have different roles during the developmental stages of D. melanoguster.DNA ligase catalyzes the formation of phosphodiester linkages between DNA chains. In eukaryotes, two forms of DNA ligase have been identified: I and 11. Because DNA ligase I is most abundant in proliferating cells, it is thought to be involved primarily in DNA replication. Likewise, the presence of DNA ligase I1 in resting cells suggests that it is involved in DNA repair. DNA ligases I and I1 are also distinguishable by their affinity for ATP, optimum ionic strength and substrate specificity [l, 21. We are interested in studying the biochemical role of the two DNA ligases in developmental stages of eukaryotes. Drosophila melanogaster was chosen for this purpose because this organism is well characterized biochemically and genetically. In a previous paper [3], we reported the detection of two DNA ligases which were separated by phosphocellulose chromatography. DNA ligase I eluted at 0.2 M KC1 from phosphocellulose and was most abundant in embryos from early stages of development. DNA ligase I1 eluted at 0.6 M KCl from phosphocellulose, was less abundant than DNA ligase I, and was present in relatively similar quantities throughout development. These enzymes appear to be similar to mammalian DNA ligases I and 11. Rabin et al. [4, 51 have also purified and characterized a DNA ligase from embryos of D . melunogaster which seems to correspond to DNA ligase 1.This paper describes the further purification and characterization of DNA ligase 11 from pupae. A comparis...

DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product

“…Since Cdc9 DNA ligase has very similar physical and biochemical properties to DNA ligase I of Drosophila (Rabin et al, 1986;Rabin & Chase, 1987) and mammalian cells (Tomkinson et al, 1990(Tomkinson et al, , 1991a, the product of the yeast CDC9 gene can be designated as S. cerevisiae DNA ligase I. Drosophila (Takahashi & Tomizawa, 1990) and mammalian cells (Tomkinson et al, 1991a; contain additional species of DNA ligase, but the cellular roles of these enzymes have not been determined. S. cerevisiae is an attractive model eukaryotic system for studying the cellular roles of multiple DNA ligases because of the potential for identifying genes which encode such enzymes and for isolating mutants.…”

Mammalian Dna Ligases

“…In all these properties, the enzyme resembles mammalian DNA ligase II. In terestingly, a small amount of an apparent "larger form of DNA ligase II" has also been observed during fractionation of Drosophila extracts (98,100) , hinting at the possibility that a third DNA ligase might be present in this organism.…”

“…A second DNA ligase with properties similar to those of mammalian DNA ligase II has been partially purified from Drosophila pupae and embryos (98,100). In common with the mammalian enzyme, Drosophila DNA ligase II is not extracted from cell nuclei with an isotonic sucrose buffer of low ionic strength, and this accounts for an early unsuccessful attempt to detect the enzyme (97).…”

Characterization of DNA Ligase from the Fungus Coprinus cinereus

“…In contrast, mammalian DNA ligase I11 is unable to perform bluntend joining even in the presence of poly(ethy1ene glycol) [22, 231. Molecular properties of pachytene DNA ligase. Since the ligase was not purified to complete homogeneity, the polypeptides with DNA ligase activity were required to be labeled with radioactive ATP (see [9,10,13,231). The purified enzyme preparation (Table 1, SDS/PAGE.…”