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 ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.
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PMID:Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro. 608 90

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.
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PMID:Cleavage of phosphorothioate-substituted DNA by restriction endonucleases. 609 46

Covalently closed-circular, superhelical SV4O DNA was used in all experiments. EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA. S1 does cleave both strands of superhelical DNA at unbasepaired sites.
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PMID:Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions. 617 3

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.
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PMID:A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase. 626 Apr 93

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.
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PMID:Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model. 626 31

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.
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PMID:Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay. 627 19

We have constructed a hybrid plasmid molecule that contains the complete coding sequences from the 26S mRNA of Semliki Forest virus. Five fragments which together covered the mRNA sequence were isolated from three original hybrid plasmids and joined together at five different restriction endonuclease cleavage sites using T4 DNA ligase.
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PMID:Construction of a hybrid plasmid molecule containing the total coding region of Semliki Forest virus 26S mRNA. 627 65

The influence of polyamines on various enzymes involved in the excision repair pathway of DNA, such as UV endonuclease, DNA polymerase I, DNA ligase and polynucleotide kinase, and two AP-endonucleases, were studied. The polymerizing activities of DNA polymerase I and polynucleotide kinase were found to be markedly affected by polyamines. In the former enzyme the effect can be attributed to the stabilization of the correct bihelical structure at the 3' end and in the latter case polyamines stabilize the polynucleotide kinase protein itself in the correct oligomeric structure. The effect of polyamines on the hydrolysis of apurinic and apyrimidinic sites in DNA and nucleosome particles were also investigated. Spermine and spermidine were found to be the most efficient polyamines in causing such hydrolysis both in the free DNA and in the nucleosome particles.
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PMID:Effect of polyamines on enzymes involved in DNA repair. 627 78

Single strand specific mung bean nuclease was used to probe for regions of altered secondary structure in supercoiled PM2 DNA. Supercoiled DNA is cleaved greater than or equal to 10,000 times faster than the relaxed topoisomer. Catalytic quantities of enzyme convert supercoiled DNA to both nicked-circular and unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. At the elevated enzyme concentrations required to cleave relaxed DNA, unit length linear DNA and smaller fragments are produced from pH 5 to 7. One nick per supercoiled DNA molecule is introduced at pH 6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snake venom phosphodiesterase selectively cleaves the strand opposite the nicks, permitting restriction endonuclease mapping. The nicks occur at three specific sites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, while a site at 0.82 is cleaved less frequently. The former sites map near one of the eight known early denaturation regions of PM2 DNA, while the latter does not. Since most early denaturation sites are not cleaved, sites other than these dA + dT-rich regions may be the preferred locations of strand unwinding and separation in supercoiled PM2 DNA.
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PMID:Action of mung bean nuclease on supercoiled PM2 DNA. 628 55

The twisting potential of DNA has been determined directly by a method that measures the cyclization probability or j-factor of EcoRI restriction fragments as a function of DNA twist. The cyclization probability is proportional to Kc, the equilibrium constant for cyclization of the restriction fragment via its cohesive ends (Shore et al., 1981). Here we vary the twist of the DNA by making small internal additions to or deletions from a 242 bp EcoRI restriction fragment. A series of 12 DNA molecules has been studied, which range in length from 237 to 254 bp. The cyclization probability is measured from the rates of covalent closure by phage T4 DNA ligase of two systems: (1) a linear restriction fragment in equilibrium with its cyclized form and (2) half molecules (cut by a blunt-end endonuclease) in equilibrium with joined half molecules. The striking result is that, in this DNA size range, the j-factor depends strongly on the fractional twist: the difference between the total helical twist and the nearest integer. Thus j depends in an oscillatory manner on DNA length between 237 and 254 bp with a period of about 10 bp. These data give the free energy of DNA twisting as a function of twist. The curve of j versus DNA length can be fitted to a harmonic twisting potential with a torsional constant of C = 2.4 X 10(-19) erg cm. This value is in reasonable agreement with different estimates of C made by Barkley & Zimm (1979: C = 1.8 X 10(-19) to 4.1 X 10(-19) erg cm) and is somewhat larger than the value obtained resulting from the kinetics of DNA twisting measured by fluorescence depolarization of ethidium intercalated into DNA (C = 1.4 X 10(-19) erg cm; Millar et al., 1982; Thomas et al., 1980) or from spin label studies (Hurley et al., 1982). Our experiments provide a direct measurement of the torsional free energy and they show that the DNA twisting potential is symmetric. Our experiments also indicate that the DNA helix is continuous, or nearly so, in a nicked circle; presumably this happens because the DNA stacking interaction maintains the double helix in register across a single-strand nick. As a consequence, the twist of a singly nicked DNA circle is integral for small (approximately equal to 250 bp) planar DNA circles and there is a change in twist upon cyclization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Energetics of DNA twisting. I. Relation between twist and cyclization probability. 631 55


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