Changes of DNA Ligases in Chick Neural Retina as a Function of Age

In the course of chick neural retina development, several forms of DNA ligase have been found. During embryonic life the major DNA ligase activity that is found at seven days is form I (8.2 S) which gradually decreases and disappears by 14 days after incubation, whereas form II (6.2 S) increases to reach a maximum at the time of hatching. Form II then decreases reaching a constant level by Day 7 and from that time new slow sedimenting forms also appear (forms III and IV). Form III (2 S) is first detectable at seven days and increases up to 90 days, whereas form IV (3 S) is the only form detected in the 17- and 18-month-old and also in the 5-year-old birds. These four forms display different elution patterns on phosphocellulose column chromatography. They also differ in their thermal stability and sensitivity towards N-ethylmaleimide.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

alpha-D-mannosidase forms in chicken liver

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.     

Developmental changes of DNA ligase during ram spermatogenesis

The molecular forms and activities of ram DNA ligase have been investigated during spermatogenesis from the stage of early round spermatids to ejaculated spermatozoa. Through germ cell maturation, two consecutive forms of the enzyme (6S and 7S) have been found. The 6S form (DNA ligase II) is observed in primary and secondary spermatocyte, as well as in round spermatids. The 7S form (DNA ligase I) is present in elongated spermatids and in the sole round cell population with spermatogonia and young primary spermatocytes. In ram germ cells, DNA ligase I and DNA ligase II appear to be respectively associated with DNA replication repair. The absence of DNA ligase II associated with the absence of DNA repair in testicular and ejaculated spermatozoa might be related to male infertility.     

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.     

In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay

We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.     

Multiple forms of oviduct progesterone receptors analyzed by ion exchange filtration and gel electrophoresis.

Resolution of the multiple forms of steroid receptors in small samples has been improved by two new techniques: preparative ion exchange filtration and electrophoresis in highly cross-linked polyacrylamide gels of varied concentration. These techniques were used in conjunction with protamine precipitation, gel filtration, and density gradient centrifugation to separate five forms of the progesterone receptor of chick oviduct cytosol. These complexes, numbered I to V in order of elution from agarose gel columns, have been characterized with respect to apparent molecular weight, shape, and relative net charge. Form I, which is eluted in the void volume after gel filtration of cytosol in hypotonic media, is heterodisperse with respect to sedimentation coefficient and electrophoretic mobility (Rf). Form I is converted to form III by KC1. Form II has the highest axial ratio and the highest Rf at pH 10.2. This 4.2S complex can be extracted from DEAE filters, but not from protamine-precipitated cytosol, by 0.3 to 0.5 M KC1. Form III is slightly smaller (3.9S) and less asymmetric than form II. It is relased from DEAE filters and protamine-precipitated cytosol by 0.15 M KC1 and displays increased Rf upon purification. Forms II and III correspond to the B and A components described by W. T. Schrader and B. W. O'Malley ((1972), J. Biol. Chem 247, 51). Form IV may result from the proteolytic cleavage of forms II and/or III. Form V is a globular polypeptide obtained in the presence of certain divalent cations. This complex has been named the "mero-receptor" since it is the smallest part or fragment of the receptor that contains the steroid-binding site.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-α forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-β, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

Multiple forms of ribonuclease II in mouse liver: relative sizes, subcellular distributions, and pH-activity profiles

Multiple forms of ribonuclease II (EC 3.1.27.5) have been resolved from extracts of crude fractions of mouse liver by ion-exchange chromatography on phosphocellulose and gel permeation chromatography. The forms are designated 6S, 6L, 5S, 5L, 4S, 4L, 3S, 3L, 2, and 1 in increasing order of apparent cationic character. The forms fall into two series of apparent molecular weight. The small series increases from molecular weight equal to 9000 for form 1 to 14,000 for form 6S. The large series increases from molecular weight equal to 22,000 for form 2 to 44,000 for form 6L. All forms have pH-activity profiles with maxima near pH 7. Activity falls to no less than 30% of this maximum at pHs 5 and 8.5. Relative to the other forms, form 1 has a higher ratio of activity in the alkaline compared with acid pH range. Form 1 is found in the cytosolic, "light" particle, and "heavy" particle fractions. The other forms are largely restricted to the heavy particle fraction. In this fraction the proportion of total activity attributable to each form generally decreases in order from form 1 down to form 6. The results are accommodated by models in which one or more gene products give rise to multiple forms of ribonuclease II by processes involving dimerization and glycosylation.     

Isolation from bovine liver mitochondria and characterization of three distinct carboxylic acid:CoA ligases with activity toward xenobiotics

A mitochondrial freeze/thaw lysate was fractionated on a DEAE-cellulose column into four distinct acyl-CoA ligase fractions. First to elute was a 50 kDa short-chain ligase that activated only short-chain fatty acids. Next to elute were three ligases that had activity toward both medium-chain fatty acids and xenobiotic carboxylic acids; these were termed xenobiotic/medium-chain ligases (X-ligases) and labeled XL-I, XL-II, and XL-III, respectively, based on order of elution. The molecular weight of X-ligases I, II, and III were ca. 55,000, 55,500 and 53,000, respectively. Form XL-III showed no pH optimum; the rate increased steadily with pH beginning from pH 7.0. XL-I and XL-II showed the same behavior with benzoate as substrate, but with medium-chain fatty acids, both forms had a pH optimum at 8.8. The three X-ligases differed in substrate specificity. XL-I was the predominant nicotinic acid activating form and had the lowest K_m for benzoate. Form XL-II was the only form with measurable salicylate activity, although it was extremely low. XL-III was the only 2,4,6,8-decatetraenoic acid activating form and also was the predominant medium-chain fatty acid-activating form. By comparison of substrate specificities, it was concluded that the two previously reported ligase preparations were mixtures of the three forms. When the ligase rates were compared to previously determined N-acyltransferase rates toward benzoyl-CoA and phenylacetyl-CoA, the data showed that ligase activities are 100-fold lower, and thus the ligase is rate limiting for the conjugation of both of these xenobiotics. © 1996 John Wiley & Sons, Inc.     

DNA ligases from rat liver. Purification and partial characterization of two molecular forms

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.     

The isolation and characterization of cyclic nucleotide phosphodiesterases from Morris hepatoma 5123tc(h) and rat liver

Four main phosphodiesterase (PDE) forms were resolved and partially purified from rat liver and Morris hepatoma 5123tc(h). The activities of the high Km cyclic nucleotide PDE (form II) in hepatoma were markedly reduced compared to liver, while the activities of the low Km cAMP PDE (form III) and low Km cyclic nucleotide PDE (form IV) in hepatoma were markedly higher than those of liver. The partially purified low Km cAMP PDE's (forms III and IV) from liver showed non-linear Lineweaver-Burk plots, whereas the same enzyme forms in hepatoma displayed linear kinetics. Activation of low Km cGMP PDE activity by calmodulin was found with form I in liver whereas in hepatoma form II was responsive to calmodulin.     

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.     

New crystal forms of glutamine synthetase and implications for the molecular structure.

New crystal forms of glutamine synthetase from Escherichia coli are reported. Two of these (II A and II B) demand that the dodecameric molecule contains a 2-fold axis of symmetry perpendicular to the apparent hexagonal face.Whereas forms II A and II B and others reported previously (I and III A) were grown from enzyme containing covalently bound AMP groups, a third new form (III C) was grown from enzyme lacking covalently bound AMP groups. Form III C is isomorphous with form III A. This demonstrates that the addition of AMP groups, which profoundly affect the catalytic and regulatory properties of glutamine synthetase, does not alter the dimensions of the molecular envelope. Thus adenylylation of the enzyme does not seem to cause a quaternary structural transition, though small changes of intensities suggest that there may be tertiary structural changes within the subunits.Other new forms include form III B, a low symmetry polymorph, closely related to form III A, and form IV, a trigonal polymorph with large asymmetric unit. All crystal forms are consistent with a symmetry of 622 for the glutamine synthetase molecule.     

Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.     

Characterization of the multiple forms of monoamine oxidase from chronic schizophrenic sera.

Either half or one hour incubation time was enough to get a constant production of benzylaldehyde and were proportional to the amount of enzyme added. The optimal temperature of MAO, I, II, III, IV are 60 degrees, 37 degrees, 60 degrees, 45 degrees, and 37 degrees C respectively, and they follow Arrhenius equation until these optimal temperatures. Each form have optimal pH depends on substrate concentration used and the buffer used. These forms were shown to be inhibited by high substrate concentration with formation of inactive enzyme-amine complex, whereas butyl- and octylamine was found to be competitive inhibitors. Isoniazid inhibit MAO II, III, IV and V forms in a non competitive fashion, whereas MAO I inhibited competitively with respect to the substrate. Semicarbazid inhibit MAO I. III, IV and V forms in a non competitive fashion, whereas MAO II inhibited competitively with respect to the substrate.     

Binding of bis-linked netropsin derivatives in the parallel-stranded hairpin form to DNA

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5'-CCTATATCC-3' in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis-diammine Pt(II)-bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5'-CCTATATCC-3' (I), 5'-CCTTAATCC-3' (II), 5'-CCTTATTCC-3' (III), 5'-CCTTTTTCC-3' (IV) and 5'-CCAATTTCC-3' (V) decreases in the order I = II > III > IV > V . The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.