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)

In this paper we describe the preparation of hybrid plasmid consisting of ColE1 DNA and DNA of R6K plasmid. ColE1 plasmid represents a circular DNA with the molecular weight of 4,2-10(6) daltons. It determines colicine synthesis E1, has relaxed control of replication and is present in the cell in several dozens copies. Transmissive plasmid B6K represents a circular DNA molecule with the molecular weight of 28-10(6) daltons. It confers the resistance to amplicillin and streptomycin and belongs to the compatibility group X. This plasmid also has relaxed control of replication (up to 30 copies per cell). Circular superhelical DNA of ColE1 was prepared after it is amplification with cloramphenicol according to Clewell by ultracentrifugation in CsCl-EtBr density gradient. The yield of superhelical ColE1 DNA in our experiments was up to 300 mg/gram cells. Circular superhelical R6K DNA was isolated from E. coli strain J53 R6K grown to stationary phase in the presence of 15 mg/ml of streptomycin. The yield of superhelical DNA was equal to about 45 mg per 1 gram cells. EcoR1 restrictase was prepared from E. coli KM182 as described by Thomas et al. From 37 grams of cells 10 ml of enzyme solution was prepared. 1 ml of this solution was able to restrict completely 2 mg of ColE1 DNA during Hu incubation for 30 degrees min at 37 degrees C. Restrictase activity was analyzed by electrophoresis of ColE1 DNA restricts in 0,7% agarose gel as described by Tanaka. DNA ligase was prepared from E. coli B. cells infected by T4 phage am81 mutant as described by Weiss. The activity of enzyme solution was equal to 90 units/ml (according to Richardson). In standard "hybridization" experiment 4 mug of ColE1 DNA and 4 mug of R6K DNA were incubated for 1 hour at 37 degrees in 0,2 ml of the solution containing 40 mM tris-HCl, pH 7,4; 10 mM MgCl2 50 mM NaCl, 10 mM mercaptoethanol and 5 mul of EcoR1 restrictase. The reaction was terminated by heating the reaction mixture to 65 degrees C for 5 min. In accordance with the results of others CoLE1 DNA gave one restrict and R6K DNA-two restricted fragments. 100 mul of the restricted mixture was chilled to 0 degrees and the following ingredients were added 10 mul of 50 mM MgCl2; 10 mul of 100 mM dithiotreitol; 10 mul of 0,5 mM ATP, 20 mul of water and 8 mul of ligase solution (90 units/mul). The mixture was incubated at 0-0,5 degrees for 53 hours, thereafter it was used for the transformation of E. coli C600 according to Tanaka et al. Several classes of hybrid molecules due to different combinations of restricted fragments of parental species were expected. These molecules had to confer the immunity to colicine E1 and to carry the defect in the gene controlling colicine synthesis since EcoR1 restrictase splits ColE1 DNA in the region of corresponding gene. In addition, certain classes of transformants with hybrid DNA molecules had to be resistant to one or both antibiotics as determined by the R6K plasmid portion...
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PMID:[Preparation of functioning recombinant (hybrid) DNA molecules in vitro (genetic engineering experiments)]. 77 38

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

The enhancement of tumor development following acute stress has been demonstrated in some animal studies. This study was designed to explore mechanisms that would account in part for the relationship between stress and tumor development at the level of DNA repair, using a rat model. Forty-four rats were given the carcinogen dimethylnitrosamine in their drinking water, and half were randomly assigned to a rotational stress condition. The levels of methyltransferase, a DNA repair enzyme induced in response to carcinogen damage, were significantly lower in spleens from the stressed animals. These data suggest that stress may impair DNA repair.
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PMID:Effects of stress on methyltransferase synthesis: an important DNA repair enzyme. 407 16

Two types of DNA-duplexes containing the repeating fragments of natural promoters have been obtained starting from synthetic oligodeoxyribonucleotides TGCATTATAA, AACTAGTT, AGTTAACT. Deca- and octanucleotides have been synthesized by solid phase method with stepwise or blockwise chain elongation. UV- and CD-spectroscopy has been used to study the physico-chemical properties of the synthetic oligonucleotides. Polycondensation of oligonucleotides induced by water-soluble carbodiimide (chemical ligation) or T4-polynucleotide ligase allowed to synthesize the promoter models. The degree of polymerization varied from 2 to 8 in case of chemical ligation and from 2 to 30 in case of enzymatic ligation. A new chain length regulation technique has been developed by means of addition of a terminator of polycondensation (unphosphorylated oligonucleotide) in the reaction mixture.
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PMID:[DNA-like duplexes containing repetitive sequences. VII. Chemico-enzymatic synthesis of polymers with fragments of natural promotors]. 670 55

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.
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PMID:Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. 767

We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0 degrees C using a water-soluble carbodiimide as condensing agent. We find that only 2 bp at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal for ligation by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.
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PMID:In vitro selection of optimal DNA substrates for ligation by a water-soluble carbodiimide. 808 82

An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.
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PMID:Two-minute miniprep method for plasmid DNA isolation. 818 27

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.
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PMID:Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV. 909 50

Chlorinated tap water often contains 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), which is a potent directly acting bacterial mutagen. We have investigated the induction of DNA damage by MX in a promyelocytic human leukaemia cell line (HL-60 cells). Exposure of HL-60 cells to 100-300 microM MX resulted in increased levels of DNA single-strand breaks and/or alkali-labile sites (SSBs) as detected by alkaline filter elution. When adding inhibitors of DNA break repair (AraC plus hydroxyurea), increased levels of DNA SSBs were observed at very low concentrations (1-3 microM) of MX, as observed by both alkaline filter elution and the single-cell gel electrophoresis assay. Increased DNA SSBs could also be observed if DNA repair inhibitors were added immediately after exposure to 10 microM MX, indicating that low concentrations of MX cause a relatively stable modification of DNA that may be recognized and incised by DNA repair enzyme activities. Further studies with DNA break repair inhibitors indicated that HL-60 cells exposed to 10 microM MX for 1 h repaired 50% of their initial DNA damage during a 2-h period and the repair appeared to be complete at 22 h. Analysis of MX-treated DNA by sequencing methods indicated that MX preferentially reacts with guanines in DNA.
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PMID:DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in HL-60 cells and purified DNA in vitro. 915 Jul 66

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.
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PMID:A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase. 1019 86


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