Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.
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PMID:A new system for studying molecular mechanisms of mutation by carcinogens. 22 5

The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.
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PMID:Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions. 133 62

The human DNA repair enzyme, methylguanine-DNA methyltransferase (MGMT, M(r) 21,000), which protects cells against the mutagenic effect of alkylating carcinogens, was found to be localized in the cell nucleus (except the nucleolus) by immunofluorescence staining using polyclonal and monoclonal antibodies. The supporting experiments came from differential staining of the MGMT-deficient (mer-) and -proficient (mer+) cells, Western blotting analysis, and specific antibody depletion studies with the immobilized fusion protein, GSTMGMT-glutathione-Sepharose. Its localization in the nucleus agrees with its biological function and possibly explains the ineffective protection of mammalian cells (mer-) transfected with the Escherichia coli MGMT genes from bifunctional alkylating agents.
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PMID:Intracellular localization of human DNA repair enzyme methylguanine-DNA methyltransferase by antibodies and its importance. 138 61

Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180 degrees out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC" (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC" can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.
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PMID:Inhibition of T4 DNA ligase activity by (+)-CC-1065: demonstration of the importance of the stiffening and winding effects of (+)-CC-1065 on DNA. 154 25

The level of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (MGMT) was examined in benign and malignant skin tumors induced with different initiating and promoting agents and from both SENCAR and Sensitive SENCAR Inbred (SSIN) mice. The MGMT levels in the tumors were approximately one-half the level observed in normal surrounding epidermis and in keratinocytes from untreated controls. In addition, a carcinoma-producing cell line, VT 17DT, derived from papillomas in SENCAR mice had no detectable MGMT activity (Mer- phenotype), whereas in the non-tumor forming line, 3PC, MGMT activity was comparable to that in papillomas. The comparatively low level of MGMT in papillomas may contribute to their ease of conversion to squamous cell carcinomas by N-ethyl-N-nitrosourea or n-methyl-N'-nitro-N-nitrosoguanidine. MGMT activity was also determined in the epidermis of non-exposed mice of various stocks and strains. Epidermal MGMT activity was similar to levels in the corresponding livers and was, in general, parallel with stock/strain susceptibility to tumor formation. This is the first report that examined MGMT activity in skin tumors and normal keratinocytes in the mice of several stocks and strains.
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PMID:O6-alkylguanine-DNA alkyltransferase activity in epidermal tumor and normal epidermal cells of mice of various stocks and strains. 163 96

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.
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PMID:DNA helicase IV from HeLa cells. 164 52

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

When animals are treated with carcinogenic agents that alkylate O6-guanine residues, the incidence of tumors in specific tissues often relates inversely to the level of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) present in the tissue. Similarly, the hypersensitivity to anticancer chloroethylnitrosoureas of some human tumor cell lines is believed to result from their deficiency in MGMT. We have undertaken a comprehensive investigation of MGMT expression in a panel of nine characterized human glioma cell lines. Methyltransferase activity determined by incubating protein extracts of these glioma lines with [3H]methylated DNA ranged from undetectable in six lines (the Mer- phenotype) to greater than 0.8 pmol/mg in two lines (U-373 MG and D-392 MG). MGMT protein was undetectable in Western blots of the Mer- cell extracts probed with specific anti-MGMT monoclonal antibodies. Consistent with these results, steady-state levels of MGMT mRNA, determined by Northern blot analysis, were detectable only in the three Mer+ glioma lines (U-373 MG, D-392 MG, D-263 MG). Southern analysis of EcoRI-digested DNA probed with MGMT cDNA revealed no amplification, rearrangement or deletions of the MGMT gene in any of the glioma cell lines. This is the first report that examines MGMT expression at the biochemical, molecular and genetic levels in a particular tumor type. These studies suggest that transcriptional regulation is the basis of the Mer- phenotype in these malignant human glioma cell lines, since no gross structural or quantitative abnormalities of the MGMT gene were seen in the phenotypically Mer- lines.
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PMID:Expression of O6-methylguanine-DNA methyltransferase in malignant human glioma cell lines. 189 34

Different biological aspects of a novel 2-chloroethyl nitrosourea derived from cysteamine, N'-(2-chloroethyl)-N-[2-(methylsulfinyl)ethyl]-N'- nitrosourea (CMSOEN2), were studied. Drug-induced cytotoxic effects, uptake kinetics, DNA damage, and O6-alkylguanine-DNA alkyltransferase activity were determined in 3 melanoma cell lines: the murine B16 and 2 human metastatic-derived cell lines (M4 Beu and M3 Dau). We found that radioactivity uptake and incorporation in acido-precipitable material was inversely proportional to cell drug viability. The highly CMSOEN2-sensitive B16 line showed the lowest total radioactivity uptake. In fact, among the melanoma cell parameters studied, 3 of them were well correlated: (a) cytotoxicity as reflected by the colony-forming assay; (b) DNA cross-link frequency estimated by the alkaline elution technique; and (c) O6-alkylguanine-DNA alkyltransferase activity (Mer phenotype), defined as the ability of cell extracts to remove O6-methylguanine from N-methyl-N-nitrosourea-alkylated DNA. The 2 human cell lines (M4 Beu and M3 Dau), the most resistant to the cytostatic drug effects, showed little or no ability to form DNA lethal cross-links. These results correspond to the higher O6-alkylguanine-DNA alkyltransferase activity found in human-derived cell lines compared with that present in murine B16 cell lines. This study confirms that the cell content in this repair DNA protein is certainly one of the important factors implicated in the variability of response to 2-chloroethyl nitrosourea treatment observed in a number of established malignant cell lines. It has been shown that pretreatment of derived cell lines with methylating agents (N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine) or O6-methylguanine used as a free base, increased cytotoxic effects of this class of anticancer agents, likely by saturating receptor sites (sulfhydryl groups) of this specific DNA repair enzyme. Nevertheless, in preliminary Phase I and II clinical trials, 2 patients who had been treated with multiple chemotherapies including alkylating agents [1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, platinum derivatives], presented complete or partial remission after CMSOEN2 treatment. Our results raise the question of the exact relation between the Mer phenotype determined in derived murine or human cultured cells and that directly observed on surgically excised tumors in cancer patients. The original Mer phenotype could be modified by cell culture conditions since it has been shown that O6-alkylguanine-DNA alkyltransferase activity is widely distributed between normal and tumoral tissues without any real difference.
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PMID:DNA damage induced by a new 2-chloroethyl nitrosourea on malignant melanoma cells. 239 61

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.
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PMID:Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps. 240 70


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