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)

This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.
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PMID:Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. 675 64

A cell-free extract from blue-green alga Anacystis nidulans contains enzymes which repair in vitro the transforming activity of gamma-irradiated Bacillus subtilis DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture, the duration of incubation and on the dose of irradiation. The repair of gamma-induced lesions is most efficient in the presence of magnesium ions, NAD and ATP. The present data indicate that the repair of transforming DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of algae.
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PMID:[In vitro repair of gamma-irradiated transforming Bacillus subtilis DNA by extracts of blue-green algae]. 680 46

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71

Replication of the lagging strand of bacteriophage T7 DNA occurs in a discontinuous fashion that requires RNA-primed DNA synthesis, the removal of the RNA primers, the replacement of the ribonucleotides with deoxyribonucleotides, and the covalent joining of adjacent DNA fragments. We have examined each of these steps as well as the whole process through the use of model substrates and partial reactions using purified proteins. Tetraribonucleotides (pppACCC or pppACCA), synthesized by the T7 gene 4 protein on single-stranded DNA, are used as primers by T7 DNA polymerase to yield RNA-terminated DNA fragments. The removal of the RNA primers is catalyzed by the 5' to 3' hydrolytic activities of either Escherichia coli DNA polymerase I or the T7 gene 6 exonuclease. The products of hydrolysis are pppApC, ATP, and nucleoside 5'-monophosphates or ATP and nucleoside 5'-monophosphates, respectively. The requirement for DNA synthesis to fill the gap between adjacent DNA fragments can be fulfilled by Form II of T7 DNA polymerase but not by Form I. DNA synthesis catalyzed by Form II of T7 DNA polymerase eliminates gaps to create a substrate for DNA ligase whereas strand displacement synthesis catalyzed by Form I creates an aberrant structure that cannot be joined. Either the host or phage DNA ligase can effect the final covalent joining. All steps in the replication of a lagging strand have been coupled in a model system that catalyzes the formation of covalently closed, circular, double-stranded DNA molecules using single-stranded viral DNA as template. A combination of four bacteriophage proteins, gene 4 protein, Form II of T7 DNA polymerase, gene 6 exonuclease, and DNA ligase, can accomplish this overall reaction.
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PMID:Bacteriophage T7 DNA replication. Synthesis of lagging strands in a reconstituted system using purified proteins. 688 17

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.
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PMID:Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro. 704 Nov 25

The capacity of eukaryotic cells to modulate the activities of DNA repair enzymes during cell proliferation was examined. Using regenerating rat liver as a model system, the specific activities of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase were determined at specific intervals after partial hepatectomy. The induction of DNA replication and the stimulation of DNA polymerase were also measured in order to relate changes in the potential for DNA repair to those observed for DNA replication. As measured in nuclear extracts, the specific activities of both the uracil DNA glycosylase and the 3-methyladenine DNA glycosylase were increased in regenerating rat liver reaching maximal levels 18--24 h after partial hepatectomy. The specific activity of each DNA repair enzyme returned to basal levels by 48 h after the hepatectomy. No increase in either enzyme activity was observed in sham operated controls. The products of the reactions were identified as 3-methyladenine or as uracil by high pressure liquid chromatography or by gel filtration on Sephadex G-10. The 2--3 fold increases in the specific activity observed for each nuclear DNA repair enzyme was comparable to the 2.7 fold increase observed for DNA polymerase activity. The stimulation of DNA repair enzymes in regenerating rat liver is a further suggestion that eukaryotic cells actively regulate excision repair pathways in the defined pattern of gene expression observed during the eukaryotic cell cycle.
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PMID:Induction of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase in regenerating rat liver. 727 38

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.
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PMID:Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase. 751 42

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.
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PMID:Detection of point mutations with a modified ligase chain reaction (Gap-LCR). 753 8

The ligation-independent cloning of PCR products (LIC-PCR) is a versatile and highly efficient cloning procedure resulting in recombinant clones only. Recombinants are generated between PCR products and a PCR-amplified vector through defined complementary single-stranded (ss) ends artificially generated with T4 DNA polymerase. This procedure does not require restriction enzymes, alkaline phosphatase, or DNA ligase. The primers used for amplification contain an additional 12-nucleotide sequence at their 5' ends that is complementary in the vector- and insert-specific primers. The (3'-->5') exonuclease activity of T4 DNA polymerase is used in combination with a predetermined dNTP (dGTP for the inserts and dCTP for the vector) to specifically remove 12 nucleotides from each 3' end of the PCR fragments. Because of the complementarity of the ends that are generated, circularization can occur between vector and insert. The recombinant molecules do not require in vitro ligation for efficient bacterial transformation. To make this technique widely applicable, we have simplified the handling of the PCR fragments prior to LIC. The PCR products do not need further purification following the T4 DNA polymerase treatment. Incubation of vector and insert PCR fragments for as little as 5 min is sufficient for a high yield of recombinants. Comparison of the transformation efficiencies using different-length LIC tails revealed that using 12-nucleotide cohesive ends produced four times more transformants than were obtained with the LIC with 10-nucleotide cohesive ends. When the LIC tails were 8 nucleotides long, no transformants were obtained. PCR fragment purification, T4 DNA polymerase treatment, and LIC is complete in < 1 hr.
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PMID:Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. 758 Sep 2

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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PMID:Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. 759 Mar 20


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