DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed, leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4°C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.DNA ligase promotes the formation of a phosphodiester bond between 3'-hydroxyl and 5'-phosphoryl termini in a double-stranded DNA molecule and its activity is essential in joining DNA chains during replication, repair and recombination processes [I]. To fulfill these different functions a unique enzyme form has been found in T4 bacteriophage [2], E. coli [3] and yeast [4, 51. In mammalian cells two enzyme forms have been reported, DNA ligases I and 11, which seem to be involved in DNA replication and in DNA repair respectively [6]. Since these two activities can be distinguished from each other by their physical properties of molecular mass and sedimentation coefficient, ligases I and I1 activities can be separated in certain conditions either after chromatography or after sedimentation in velocity gradients of cellular extracts [7 -101. In fact after gel filtration analysis or velocity gradient sedimentations DNA ligase I has been described as a large enzyme with an apparent molecular mass ranging from 175 kDa to 220 kDa and a sedimentation coefficient of about 8 S, whereas ligase I1 has been described as a smaller enzyme of about 85 kDa and 4.5 S [6,7,10]. More recently it has been shown that, after NaDodS04/polyacrylamide gel electrophoresis, purified DNA ligases I and I1 from calf thymus present a single polypeptide with a molecular mass of 130 kDa and 67 kDa respectively [II, 121. DNA ligase I is supposed to be
A phylogenetic survey for the poly(ADP-ribose)polyrase has been conducted by analyzing enzyme activity in various organisms and determining the structure of the catalytic peptides by renaturation of functional activities of the enzyme in situ after electrophoresis in denaturating conditions (activity gel).The enzyme is widely distributed in cells from all different classes of vertebrates, from arthropods, mollusks and plant cells but could not be detected in echinoderms, nematodes, platyhelminthes, thallophytes (including yeast) and bacteria. The presence on activity gels of a catalytic peptide with M , = 115000-120000 was demonstrated in vertebrates, arthropods and mollusks but no activity bands were recovered in many lower eukaryotes, in plant cells and bacteria. By using an immunological procedure that used an antiserum against homogeneous calf thymus poly(ADP4bose) polymerase, common immunoreactive peptides were visualized in mammals, avians, reptiles, amphibians and fishes, while lacking in non-vertebrate organisms.Our results indicate that the structure of poly(ADP-ribose) polymerase is conserved down to the mollusks suggesting its important role for DNA metabolism of multicellular organisms.The enzyme poly(ADP-ribose) polymerase utilizes NAD as substrate for transferring ADP-ribose moieties to nuclear acceptor proteins (histones, non-histone proteins) and the enzyme itself [l]. The reaction of ADP-ribosylation is extended to form a polymer of different length, with a linear (21 or branched [3] structure. The role of this enzyme is not well established. The poly(ADP-ribose) polymerase seems to be involved in the modification of the chromatin structure and consequently in the main processes of DNA function [l, 4, 51.The poly(ADP-ribose) polymerase is known to be localized in the nucleus but its distribution among eukaryotic cells has not been well studied. It is reported to be present in almost all nucleated cells, although terminally differentiated cells lack the enzyme activity [l]. It has been identified also in the lower eukaryotes Physarum polycephalum [6], Dictyostelium discoideum [7], Tetrahymena pyriforrnis [8] and Plasmodium malariae [9] whereas evidence for its presence in yeast is still'controversial [l, 10,111. Recently the enzyme has been reported also in the dinoflagellates [12].Phylogenetic investigations can be very useful for understanding the physiological role of an enzyme, its degree of conservation, the relationship between structure and function, the identification of possible precursors and catalytic subunits [13, 141. The principal deterrents to these kinds of studies Correspondence to U. Bertdzzoni, Istituto di Genetica Biochimica ed Evoluzionistica, C.N.R., Via Abbiategrasso, 207, 1-27100 Pavia, ItalyAbbreviations. CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; PhMeS02F, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; Cl,AcOH, trichloroacetic acid ; TEMED, N,N,N"'-tetramethylethylene diamine.Enzyme. Poly(ADP-ribose) polymerase, NAD: protein (ADP-n...
The inhibitors of the nuclear enzyme ADP-ribosyl transferase (ADPRT) had been shown to block the stimulation of quiescent lymphocytes with mitogens suggesting the involvement of the enzyme in the control of gene expression and cell differentiation. By means of the activity-gel assay we have analysed the intensity and the molecular mass of the catalytic bands of the enzyme at early and late times after stimulation of human lymphocytes by phytohemagglutinin. We observed that the increase in the activity of ADPRT is concurrent with the onset of DNA synthesis and is maintained for up to 10 days after lymphocyte stimulation, when DNA replication is over but the capacity to perform repair synthesis is still elevated. The analysis of ADPRT in stimulated lymphocytes by Western blots indicated that the increase in enzyme activity is due to the de novo synthesis of enzyme protein. The response of ADPRT to the treatment of human lymphocytes with DNA-damaging agents was studied at various dose-ranges, using the activity-gel technique. The results obtained indicate that dimethyl sulfate is 10 times as active as methyl methane sulfonate in stimulating ADPRT activity and that, at very high doses, the activity band of the enzyme tends to disappear. Very similar observations were obtained when Chinese hamster ovary cells were treated with the same agents, although the concentrations of the mutagens eliciting maximal ADPRT activation were 10 times higher than in human lymphocytes. When analysed by Western blots, no significant difference of the protein band of the enzyme was observed in comparing control and treated cells. This suggests that the activity-gel system can detect two different phenomena: the increase in enzyme protein, as in the case of stimulated lymphocytes, and the enzyme-activating effect of DNA-damaging agents, which occurs without changing the number of enzyme molecules. Of particular interest is the observation that mitomycin C is capable of activating ADPRT in human lymphocytes, thus suggesting that cross-linking agents are involved in promoting ADP-ribosylation reactions. We have also analysed the variations of the enzyme throughout the cell cycle in HeLa cells synchronized in S phase or in mitosis. No significant changes in the levels of the enzyme activity were revealed by the activity-gel assay during the progression of the cycle, although an overall increase of active polypeptides of larger size in concomitance with the S period was observed.
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