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)

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.
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PMID:Site-directed modification of DNA duplexes by chemical ligation. 337 71

We have investigated the fate of the yeast 2 micron DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.
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PMID:Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant. 352 47

Chemical and enzymatic ligation between the 5'-terminal phosphate of one oligonucleotide and the 3'-terminal 2',3'-cis-diol group of the other oligonucleotide on a complementary template was studied. Carbodiimide, imidazolide and N-hydroxybenzotriazole ester methods were used for chemical activation of the phosphate group, and T4 DNA ligase for enzymatic ligation. All the chemical activation methods produced 3',5'- and 2',5'-phosphodiester bonds (40-45 and 55-60%, resp.), whereas enzymatic ligation gave the product only with 3',5'-phosphodiester bond.
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PMID:[Chemical reactions in double-stranded nucleic acids. The nature of the bond formed during chemical ligation using a cis-diol group]. 367 53

Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.
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PMID:Nicks 3' or 5' to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase. 368 72

A Chinese hamster cell mutant (XR-1) was previously described that is extremely deficient in the repair of double-strand DNA breaks produced by gamma-irradiation during the sensitive G1--early-S period and somewhat deficient in repair of gamma-ray-induced single-strand DNA breaks. To determine whether a deficiency in DNA ligase activity might underlie the biochemical defect, protein extracts from mutant and parental cells were examined for their ability to ligate single- and double-strand breaks in DNA. The kinetics of ligation of single 5'-phosphate-3'-hydroxyl breaks in double-stranded DNA were the same in protein extracts from both cells. After separation of protein extracts by gel-filtration chromatography, the percentage of activity in the large and small molecular forms of DNA ligase was also similar in the two cells. Finally, protein extracts prepared from exponentially growing or G1-synchronized mutant and parental cells were equal in their ability to ligate blunt-end DNA substrates. These data suggest that a deficiency in DNA ligase is not the cause of the repair defect in the XR-1 mutant cell.
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PMID:Normal DNA ligase activity in a gamma-ray-sensitive Chinese hamster mutant. 379 64

The polynucleotide ligase isolated from T4-infected Escherichia coli was previously shown to bring about repair of breaks in the single strands of bihelical DNA. The present work shows that the enzyme can also catalyze the joining of DNA duplexes at their base-paired ends. This novel reaction occurs when the deoxynucleoside at a 5'-end carries a phosphate group and the complementary deoxynucleoside opposite to it carries a 3'-hydroxyl group. The consequence is the lengthening of the original duplex to form dimers or oligomers depending upon whether one or both ends are base-paired.
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PMID:Studies on polynucleotides, C. A novel joining reaction catalyzed by the T4-polynucleotide ligase. 527 71

The 5' AP endodeoxyribonucleases hydrolyze the phosphodiester bond 5' to AP (apurinic or apyrimidinic) sites in double-stranded DNA leaving 3'-OH and 5'-phosphate ends. These nicks are sealed by T4 DNA ligase although the 5'-phosphate end belongs to a base-free deoxyribose.
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PMID:T4 DNA ligase can seal a nick in double-stranded DNA limited by a 5'-phosphorylated base-free deoxyribose residue. 622 66

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.
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PMID:Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks. 636 Oct 28

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.
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PMID:Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V. 684 90

Kinetic analysis of the reaction catalyzed by calf thymus DNA ligase (EC 6.5.1.1) has been carried out using [5'-32P]nicked DNA as substrate. The results of initial velocity and product inhibition studies indicate that the ligase reaction is likely to proceed through the 'uni-uni uni-bi ping-pong' mechanism. The order of substrate addition and product release is as follows: ATP, PPi, nicked DNA, sealed DNA and 5'-AMP. The true Km values for ATP and for nicked DNA (5'-phosphoryl ends) were 2 microM and 0.11 microM, respectively. The turnover number was estimated to be 7 sealing events per min. dATP was an inhibitor competitive with ATP (Ki = 25 microM). The addition of 0.5 mM spermine or 5 mM spermidine resulted in an increase in the apparent Km for nicked DNA as well as in the apparent V, whereas 0.1 M KCl increased only the apparent Km for nicked DNA. Neither polyamine nor KCl affected the apparent Km for ATP. The ligase reaction was not significantly affected by aphidicolin and various phosphate compounds tested.
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PMID:Kinetic studies on the reaction catalyzed by DNA ligase from calf thymus. 688 74


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