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 DNA kinase has been partially purified from rat liver nuclei by a procedure which also yields DNA ligase. The kinase uses ATP to phosphorylate specifically the 5'-hydroxyl termini of oligodeoxynucleotides and of single- or double-stranded DNA, yielding 5'-phosphate termini and ADP. The kinase is inactive on RNA, or on oligodeoxynucleotides of chain length less than approximately 10 to 12 residues. The kinase requires a divalent cation (Mg2+, Mn2+, Co2+, Zn2+, Ni2+, or Ca2+) for activity and has an acidic pH optimum. It is inhibited by a variety of nucleotides as well as by very low levels of inorganic and organic sulfate compounds and sulfate analogues. The molecular weight of the kinase is estimated to be 8 times 10(4) from gel filtration.
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PMID:A deoxyribonucleic acid kinase from nuclei of rat liver. Purification and properties. 0 4

Neocarzionstatin (NCS)-induced strand breakage of DNA generates nonfunctional binding sites for the E. coli DNA polymerase I. Treatment of the NCS-nicked DNA with alkaline phosphatase at 65 degrees C prior to the polymerase reaction results in 60-100-fold stimulation of dTMP incorporation whereas in a control not treated with the drug there is only a 2-fold increase. Sites of strand scission on the NCS-treated DNA bear phosphate at the 3' termini. This conclusion is supported by the kinetics of release of inorganic phosphate from NCS-cut DNA by exonuclease III. Since our earlier work has shown that virtually all the 5' ends of the nicks caused by NCS bear phosphomonoester groupings, the 3'- and 5'- phosphoryl termini could be quantitated using alkaline phosphatase and exonuclease III. Over a wide range of drug levels the amount of inorganic phosphate released by alkaline phosphatase is approximately twice as much as that removed by exonuclease III, indicating the presence of equal amounts of 3'- and 5'- phosphoryl termini. This, taken together with other previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.
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PMID:Gaps in DNA induced by neocarzinostatin bear 3'- and 5'-phosphoryl termini. 14 15

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end. 17 59

The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.
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PMID:Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 9. Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94. 76 34

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.
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PMID:Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. 77 32

Experiments with the Neurospora crassa single strand-specific endonuclease have provided evidence for the existence of regions of partially single-stranded character in covalently closed superhelical replicative form DNA of phiX174. The nuclease converts the superhelical molecules to either singly hit relaxed circular or doubly hit linear molecules. We show that the initial cleavage of phiX174 superhelical DNA is a "nick" bounded by a 5'-phosphate and a 3'-hydroxyl; no nucleotides are excised as evidenced by the ability of T4-polynucleotide ligase to reform the phosphodiester bond. The nick can be found in either strand of the double-stranded DNA and is either randomly distributed or at least can be found at any one of many possible locations in the genome. Thus, the regions in phiX174 superhelical molecules that are sensitive to the N. crassa nuclease do not occur at highly specific sites in the genome.
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PMID:Site of cleavage of superhelical phiX174 replicative form DNA by the single strand-specific Neurospora crassa endonuclease. 94 17

An endonuclease from Escherichia coli which acts specificially upon UV-irradiated DNA (correndonuclease II) and is absent from the uvrA and uvrB mutants has been isolated and partially chacterized. The enzyme is present in normal amounts in the urvC mutant. It elutes from phosphocellulose at about 0.25 M potassium phosphate (pH 7.5) and passes through dialysis tubing. The enzyme binds tightly to UV-irradiated DNA but does not bind to unirradiated DNA. The enzyme incises irradiated DNA to the 5' side of a pyrimidine dimer and leaves a 5'-phosphoryl terminus which can be resealed with polynucleotide ligase. The Km of the enzyme is about 1.5 X 10(-8) M dimers. Endonucleolytic activity of the enzyme is inhibited by caffeine with a KI of about 10mM.
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PMID:The Escherichia coli UV endonuclease (correndonuclease II). 110 24

In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.
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PMID:The synthesis of a DNA duplex corresponding to the icosanucleotide sequence at the 5' end of messenger RNA from the gene N of bacteriophage lambda. 114 Dec 42

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 crystal structure of the DNA repair enzyme endonuclease III, which recognizes and cleaves DNA at damaged bases, has been solved to 2.0 angstrom resolution with an R factor of 0.185. This iron-sulfur [4Fe-4S] enzyme is elongated and bilobal with a deep cleft separating two similarly sized domains: a novel, sequence-continuous, six-helix domain (residues 22 to 132) and a Greek-key, four-helix domain formed by the amino-terminal and three carboxyl-terminal helices (residues 1 to 21 and 133 to 211) together with the [4Fe-4S] cluster. The cluster is bound entirely within the carboxyl-terminal loop with a ligation pattern (Cys-X6-Cys-X2-Cys-X5-Cys) distinct from all other known [4Fe-4S] proteins. Sequence conservation and the positive electrostatic potential of conserved regions identify a surface suitable for binding duplex B-DNA across the long axis of the enzyme, matching a 46 angstrom length of protected DNA. The primary role of the [4Fe-4S] cluster appears to involve positioning conserved basic residues for interaction with the DNA phosphate backbone. The crystallographically identified inhibitor binding region, which recognizes the damaged base thymine glycol, is a seven-residue beta-hairpin (residues 113 to 119). Location and side chain orientation at the base of the inhibitor binding site implicate Glu112 in the N-glycosylase mechanism and Lys120 in the beta-elimination mechanism. Overall, the structure reveals an unusual fold and a new biological function for [4Fe-4S] clusters and provides a structural basis for studying recognition of damaged DNA and the N-glycosylase and apurinic/apyrimidinic-lyase mechanisms.
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PMID:Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III. 141 36


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