DNA ligase I is dephosphorylated during the execution step of etoposide-induced apoptosis.
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Antineoplastic Agents, Phytogenic↗
Biomedical subjects
A Montecucco
Publications and source records attributed to A Montecucco.
Etoposide induces the dispersal of DNA ligase I from replication factories.
In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.
Antineoplastic Agents, Phytogenic↗
Pharmacological and molecular evidence for dopamine D(1) receptor expression by striatal astrocytes in culture.
The neurotransmitter dopamine (DA) at a 10 microM concentration elicited a stimulation of intracellular cyclic AMP (cAMP) accumulation in cultured astrocytes derived from embryonic rat striatum. This accumulation was partially blocked by the beta-adrenergic receptors antagonist propranolol, mimicked by the D(1) agonist SKF 38393 and by the mixed D(1)/D(2) agonist apomorphine. A regional heterogeneity in the magnitude of dopamine-induced cAMP accumulation was observed in cultured astrocytes obtained from different brain areas. The maximum effect was observed in striatal astrocytes, a lower effect in cortical astrocytes, and no increase was detected in cerebellar astrocytes. Reverse transcription-polymerase chain reaction (RT-PCR) coupled to Southern blot hybridization demonstrated that striatal astrocytes express only D(1) receptor mRNA and Western blot analysis confirmed the expression of the D(1) receptor protein in striatal astrocytes. In contrast to what found in neurons, the D(1)-dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D(1) receptors or the activation of adenylyl cyclase by forskolin led to an increase of cytosolic and nuclear protein kinase A (PKA) catalytic activity. The presence of dopamine D(1) receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes.
Animals↗
The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I.
The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After dephosphorylation in early G(1), the level of Ser66 phosphorylation is minimal in G(1), increases progressively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G(1) and S phase but not in G(2)/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.
3T3 Cells↗
L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?
We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.
Adenosine Monophosphate↗
DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.
In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.
Amino Acid Sequence↗
Potentiation of dopamine-induced cAMP formation by group I metabotropic glutamate receptors via protein kinase C in cultured striatal neurons.
Metabotropic glutamate receptors have been shown to potentiate the cyclic adenosine monophosphate (cAMP) formation induced by activation of several receptors linked to adenylyl cyclase via Gs-protein. Here we show that, in primary cultures of striatal neurons, group I metabotropic receptors potentiate the cAMP formation induced by activation of D1-like dopamine receptors. Reverse transcription associated with polymerase chain reaction revealed that mGluR5, mGluR3, mGluR4 and mGluR7 are present in striatal cell cultures. The potentiation of cAMP formation is induced by the selective group I mGluRs agonist (S)-3,5-dihydroxyphenylglycine and by other non-selective mGluRs agonists with a typical group I-like pharmacology (quisqualate > ibotenate > 1-aminocyclopentane-1,3-dicarboxylic acid). The rank order potency of mGluRs agonists in potentiating cAMP formation correlates with their ability to induce inositol phosphates production; the potentiation of cAMP formation and the inositol phosphates production are blocked by the group I mGluRs antagonists (S)-4-carboxyphenylglycine and are not affected by group II antagonist 2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine or group III antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid. The potentiating mechanism involves the activation of protein kinase C, being mimicked by phorbol-12-myristate-13acetate and blocked by the specific protein kinase C inhibitors bisindolylmaleimide I and chelerythrine or by protein kinase C downregulation. Our results indicate that this interaction could have a functional importance in modulating the cAMP-dependent transmission in the striatum.
Animals↗
Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.
ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.
Adenosine Monophosphate↗
Characterization of human DNA ligase I expressed in a baculovirus-insect cell system.
The baculovirus expression system was used to overexpress human DNA ligase I (hLig I). Approx. 2 mg of recombinant hLig I was produced per 10(8) Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active recombinant hLig I was 48 h post-infection. Lig I activity was demonstrated by auto-adenylating, polynucleotide joining and DNA relaxation assays. The baculovirus system has the advantage over previously described methods for producing hLig I of generating large amounts of a full-length active protein.
Animals↗
The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication.
DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.
3T3 Cells↗
Late induction of human DNA ligase I after UV-C irradiation.
We have studied the regulation of DNA ligase I gene expression in UV-C irradiated human primary fibroblasts. An increase of approximately 6-fold both in DNA ligase I messenger and activity levels was observed 24 h after UV treatment, when nucleotide excision repair (NER) is no longer operating. DNA ligase I induction is serum-independent and is controlled mainly by the steady-state level of its mRNA. The activation is a function of the UV dose and occurs at lower doses in cells showing UV hypersensitivity. No increase in replicative DNA polymerase alpha activity was found, indicating that UV induction of DNA ligase I occurs through a pathway that differs from the one causing activation of the replication machinery. These data suggest that DNA ligase I induction could be linked to the repair of DNA damage not removed by NER.
Cells, Cultured↗
Genetic mapping and expression analysis of the murine DNA ligase I gene.
We mapped the murine DNA ligase I gene (Lig1) in the mouse genome by using a mapping panel from an interspecific cross. Lig1 mapped to a centromeric part of chromosome 7, a region homologous to human chromosome 19q, where the human homologue LIG1 was localized. In addition, Lig1 expression was analyzed during the course of mouse liver-cell regeneration induced by partial hepatectomy, necrogenic doses of carbon tetrachloride, or the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. The results demonstrate that Lig1 is expressed in the liver during active cell proliferation.
Animals↗
Cloning and sequence analysis of a cDNA coding for the murine DNA ligase I enzyme.
A complementary DNA (2961 bp) containing the complete coding sequence for murine DNA ligase I was isolated from a mouse fibroblast cDNA library using a cDNA encoding the human protein as a probe. An open reading frame of 2748 bp, encoding a protein of 916 amino acids (aa), was identified. Northern blot analysis of total RNA extracted from mouse fibroblasts showed a single band with a mobility corresponding to a size of 3.2 kb whose level increases upon serum stimulation of quiescent mouse NIH-3T3 cells. Alignment of the murine and human deduced aa sequences showed an overall 83% identity, that rises to 91% if only the sequence on the C-terminal portion of the protein containing the active site is considered.
3T3 Cells↗
Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions.
Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.
Adenosine Monophosphate↗
Lack of discrimination between DNA ligases I and III by two classes of inhibitors, anthracyclines and distamycins.
We have measured the effects of eight distamycin and two anthracycline derivatives on polynucleotide joining and self-adenylating activities of human DNA ligase I and rat DNA ligases I and III. All test drugs show good inhibitory activity against the three enzymes in the poly[d(A-T)] joining assay. Several distamycins also inhibit the DNA-independent self-adenylation reaction catalysed by the human enzyme and, to a lesser extent, by rat DNA ligases. These results confirm that anthracyclines and distamycins express their inhibitory action against DNA joining activities mainly via specific interactions with the substrate, and suggest that the three test DNA ligases utilize similar, if not identical, mechanisms of recognition and interaction with DNA-drug complexes. Our findings also indicate that distamycins have a greater affinity for human DNA ligase I than for rat enzymes, suggesting that, in this respect, rat DNA ligase I is more similar to rat DNA ligase III than to human DNA ligase I.
Animals↗
DNA ligase I gene expression during differentiation and cell proliferation.
We have studied the regulation of mammalian DNA ligase I gene by using a cDNA probe in Northern blot experiments with RNA extracted from several cell types in different growth conditions. DNA ligase I mRNA is detected in all analysed cell systems, regardless of their proliferation state, including mature rat neurons. A significant increase in DNA ligase I mRNA level is observed when cells are induced to proliferate, in agreement with the raise of DNA joining activity found in the same cell systems. The increase parallels the start of DNA synthesis, but the messenger remains at high level beyond the end of the S phase and is detected also in the presence of aphidicolin. A decrease in DNA ligase I mRNA is observed in HL-60 and NIH-3T3 cells after differentiation. The high stability of DNA ligase I mRNA in both resting and proliferating human fibroblasts suggests a cell proliferation dependent rate of transcription. On the other hand the presence of a basal level of DNA ligase I in nondividing cells, strongly suggests an involvement of this enzyme in DNA repair. This conclusion is supported by a threefold increase in DNA ligase I observed 24 h after UV irradiation of human confluent primary fibroblasts.
3T3 Cells↗
Rat liver DNA ligases. Catalytic properties of a novel form of DNA ligase.
A novel form of rat liver DNA ligase (molecular mass 100 kDa) can be differentiated from DNA ligase I by several biochemical parameters. It is a more heat-labile enzyme and unable to join blunt-ended DNA, even in the presence of poly(ethylene glycol) concentrations which stimulate such joining by DNA ligase I and T4 DNA ligase. It also lacks the AMP-dependent nicking/closing reaction, which is a property of all other DNA ligases tested so far, including DNA ligase I from rat liver. Both rat liver DNA ligases were inhibited by deoxyadenosinetriphosphate, however this inhibition was competitive with respect to ATP, for DNA ligase I (Ki 22 microM) and non-competitive for the 100-kDa DNA ligase (Ki 170 microM). These results support the idea that, when compared with other DNA ligases, the novel form of DNA ligase has a unique AMP-binding site, may have an absolute requirement for single-strand breaks and, furthermore, may have an altered reaction mechanism to that which is conserved from bacteriophage to mammalian DNA ligase I.
Adenosine Monophosphate↗
DNA binding properties of FCE24517, an electrophilic distamycin analogue.
The distamycin derivative FCE24517 binds both reversibly and irreversibly to DNA. At 37 degrees C, the drug originates reversible complexes that are strong enough to survive to the electrophoretic separation of the substrate. These complexes slowly evolve to covalent adducts (10(-4) adducts/bp/h) that eventually degenerate to single-strand breaks (1.5 x 10(-5) nicks/bp/h). The site of attack by the drug can be any base in the vicinity of AT-rich regions of the double helix. Rapidly reassociating duplex DNA molecules, indicative of the presence of cross-links, are observed only upon boiling of DNA with FCE24517. While the low rates of formation of covalent adducts and DNA breaks could be relevant for the long-term biological effects of FCE24517, the specific formation of strong but still reversible complexes with DNA could be matched to the drastic and sudden reduction of thymidine incorporation induced by this electrophilic distamycin.
Antineoplastic Agents↗