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

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
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PMID:Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis. 200 53

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
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PMID:Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C. 204 Jun 2

Immunoaffinity-purified DNA-polymerase-alpha--DNA-primase complex from calf thymus was phosphorylated in vitro by highly purified casein kinase II from the same tissue. Specific phosphorylation of the DNA-polymerizing alpha subunit and the primase-associated gamma subunit was observed. About 1 mol phosphate/mol polymerase--primase was incorporated. Despite this effect, neither the DNA polymerase nor the DNA primase activity were changed after phosphorylation by casein kinase II. Furthermore, dephosphorylation of polymerase--primase with alkaline phosphatase did not change the polymerase or the primase activity to a significant extent. Moreover, both alkaline phosphatase and casein kinase II had no effect on the processivity of DNA synthesis and on the lengths and amounts of primers formed by the DNA primase. Because DNA polymerase alpha maintained all its basic properties even after extensive treatment with alkaline phosphatase, it is unlikely that phosphorylation has a direct influence on the activities of the DNA-polymerase-alpha--DNA-primase complex. The possible influence of post-translational phosphorylation on the formation of a complex of polymerase alpha and its accessory proteins is discussed.
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PMID:Casein kinase II phosphorylates DNA-polymerase-alpha--DNA-primase without affecting its basic enzymic properties. 222 36

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
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PMID:Ligation-independent cloning of PCR products (LIC-PCR). 223 90

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.
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PMID:Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity. 241 27

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.
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PMID:An alternative nonradioactive method for labeling DNA using biotin. 252 11

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.
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PMID:Properties of a DNA repair endonuclease from mouse plasmacytoma cells. 258 76

The direct identification of enterotoxigenic Escherichia coli from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying the heat-labile toxin (LT) gene. Two synthetic primers, each of which was 20 bases in length, were used with the thermostable DNA polymerase from Thermus aquaticus to amplify the LT gene. The amplified PCR products were detected by either gel electrophoresis or hybridization to a 24-base synthetic oligonucleotide probe conjugated to alkaline phosphatase. The PCR method detected LT-positive bacteria but did not react with E. coli producing the heat-stable toxin, enteroinvasive E. coli, Salmonella typhi, Salmonella typhimurium, or Shigella dysenteriae. By the PCR method, a single bacterium could be detected following 30 cycles of amplification. The T. aquaticus DNA polymerase was inhibited by more than 10(3) organisms in the amplification reaction mixture. A group of 40 clinical specimens consisting of 16 LT bioassay-positive and 24 LT bioassay-negative stool specimens were tested by PCR for the presence of toxigenic E. coli. The total DNA from 100 microliters of stool specimen was extracted and partially purified with a commercially available ion-exchange column. All 16 of the bioassay-positive stool specimens were positive by PCR. In addition, one stool specimen which was bioassay negative for LT but positive for LT in a previous hybridization assay with a different LT probe was also positive by PCR. This may indicate that the LT gene is present but either is not expressed or is expressed below detectable levels. Amplification of specific DNA sequences by PCR provides a highly sensitive and specific tool for the detection of pathogenic microorganisms directly from clinical specimens without the need for prior isolation. This technique may find wide application in the detection of other organisms in addition to enterotoxigenic E.coli.
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PMID:Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. 264 92

Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.
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PMID:Non-radioactive hybridization probes prepared using M13 phage vector and the universal sequencing primer. 267 2

Recent studies with crude or partially purified cell extracts have suggested that DNA polymerase alpha activity may be regulated by enzymatic phosphorylation. To further investigate these findings, we have examined the effects of protein kinases and phosphatases on highly purified DNA polymerase alpha from mouse cells. Incubation of DNA polymerase alpha with a variety of protein kinases, including protein kinase C, had no effect on polymerase activity. In addition, treatment of the polymerase with soluble calf intestinal alkaline phosphatase had no effect on DNA polymerase alpha activity, further indicating that phosphorylation does not have a direct role in modulating polymerase activity. In contrast, incubation of DNA polymerase alpha with calf intestinal alkaline phosphatase crosslinked to agarose beads resulted in a time dependent disappearance of polymerase activity. This loss of DNA polymerase activity was dependent on phosphatase activity, as the alkaline phosphatase inhibitors, potassium phosphate or levamisole, prevented the loss of polymerase activity in the presence of the beaded phosphatase. The loss of DNA polymerase alpha activity following beaded phosphatase treatment was not a general phenomena as the large fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase or mouse primase were not affected by similar treatment. The decreased DNA polymerase activity following incubation with phosphatase beads correlated with the binding of the DNA polymerase polypeptides, p185 and p68, to the agarose beads and this binding could not be reversed by either 150 mM potassium chloride or sodium sulfate. The binding of the polymerase to the agarose beads was dependent on the phosphatase activity, as the polymerase could be first treated with soluble calf intestinal phosphatase and subsequently bound to added Sepharose 4B beads. Surprisingly, Sepharose CL4B, a highly desulfated agarose preparation, did not bind the phosphatase-treated polymerase suggesting that sulfated polysaccharides are required for polymerase binding. The physiological correlate of this binding is unknown, but it has been reported that sulfated polysaccharides exist in a variety of intracellular compartments. It would be interesting to speculate that phosphorylation controls the intracellular compartmentalization of DNA polymerase alpha.
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PMID:DNA polymerase alpha activity is not affected by protein kinases or alkaline phosphatase. 293 May 69


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