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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266-276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187-197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.
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PMID:Replication of the linear mitochondrial DNA of Tetrahymena pyriformis. 11 Mar 48

The nucleotide sequence around the origin of replication in DNA of mouse polyoma virus was determined by 32P labeling of the 3' terminus of the Hap II-5/Alu I-1 DNA fragment, with the use of DNA polymerase. The result coincided with our previous report on the 32P labeling, with the use of polynucleotide kinase, of the 5' terminus of the Hap II-5/Hha I-1 DNA fragment, which corresponds to the large part of the present fragment, Hap II-5/Alu I-1. A symmetrical (A+T)-rich region containing a five-A stretch (or a five-T stretch) was flanked by two small regions with a 2-fold rotational axis of symmetry. On comparison of the sequence near the replication origin of polyoma DNA with that in the corresponding region of simian virus 40 DNA, which was included in the EcoRI-G fragment sequenced by Weissman's group (Subramanian K.N., Dahr, R. & Weissman, S. M. (1977) J. Biol. Chem. 252, 355--367), a considerable similarity was detected. Several possible common sequences for important biological activities such as the starting of DNA replication and RNA synthesis were suggested.
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PMID:Similarity of nucleotide sequences around the origin of DNA replication in mouse polyoma virus and simian virus 40. 20 28

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.
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PMID:Site-specific cleavage of DNA by E. coli DNA gyrase. 37 3

We describe a method leading to the formation of closed circles of rDNA starting from total DNA of Xenopus laevis. Linear DNA molecules were digested with exonuclease 3 and self-annealed. Open circles were enriched and covalently closed by the simultaneous use of polynucleotide kinase, DNA polymerase and polynucleotide ligase. Closed circles of rDNA1 were shown to be alkali-resistant, to have higher density than linear molecules in cesium chloride density gradients containing ethydium bromide, and to have the sedimentation constant expected for a single repeat unit of rDNA comprehensive of its spacer.
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PMID:Preparation and isolation of covalently closed circular rDNA molecules from DNA of Xenopus laevis. 67 51

Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common.
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PMID:Sequence analysis of the nicks and termini of bacteriophage T5 DNA. 86 38

The location of the protein in the open circular DNA form of the ColE1 DNA-protein relaxation complex, induced by treatment with sodium dodecyl sulfate, has been studied using several enzymes of DNA metabolism. Escherichia coli exonucleases I and III are able to degrade extensively the nicked strand of the relaxed complex from the 3' end. DNA polymerase I can initiate synthesis using the relaxed complex as template-primer and specifically extends the 3' end of the nicked strand. The 5' end of the sodium dodecyl sulfate-relaxed complex, however, is blocked to the 5'-3' hydrolitic action T7 exonuclease. This block remains after trypsin treatment of the sodium dodecyl sulfate-relaxed complex but is removed by Pronase treatment. T4 DNA ligase is unable to seal either the sodium dodecyl sulfate-relaxed complex or the Pronase-treated relaxed complex even after pretreatment of the relaxed complex with T4 DNA polymerase and polynucleotide kinase. However, pretreatment with DNA polymerase I and the four deoxyribonucleoside triphosphates facilitates ligase closure of the Pronase-treated relaxed complex but not the sodium dodecyl sulfate-relaxed complex. These studies indicate that the protein in the relaxed ColE1 complex is located at or near the 5' end of the nicked strand.
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PMID:Relaxation complexes of poasmid DNA and protein. III. Association of protein with the 5' terminus of the broken DNA strand in the relaxed complex of plasmid ColE1. 110 45

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.
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PMID:Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III. 127 22

A simple method has been developed for the isolation of DNA from agarose gels. Centrifugation in a low-cost device for 45 s at low speed provides a high yield of DNA suitable for further manipulation by restriction enzymes, T4 DNA ligase, Taq polymerase, Klenow fragment, and T4 polynucleotide kinase, and also for sequencing. In contrast, centrifugation for greater than 1 min leads to coelution of substances that inhibit DNA ligase.
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PMID:Optimized centrifugation for rapid elution of DNA from agarose gels. 135 95

A highly efficient, non-labor-intensive method for cloning DNA fragments produced by PCR amplification was used to carry out a rapid survey of potential point mutations in integrin alpha 6 cDNA from 17 different cell-type sources. The method includes glass powder purification of the PCR reaction mixture, followed by simultaneous treatment with T4 polynucleotide kinase and DNA polymerase I, and another glass powder purification. Sequences from multiple subclones of each cell type were readily generated, aligned and checked for mismatches. Several commonly used alternative procedures were compared for cloning efficiency and size-fidelity of inserted DNA fragments.
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PMID:An efficient and reliable method for cloning PCR-amplification products: a survey of point mutations in integrin cDNA. 147 31

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.
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PMID:Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin. 171 Sep


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