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)

A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.
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PMID:Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae. 4 8

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.
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PMID:Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells. 4 29

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.
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PMID:Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns. 4 87

Nonionic detergents stimulate purified RNA-directed DNA polymerase (reverse transcriptase) activity from various RNA tumor viruses ranging from avian to primate species. The stimulatory effect of the nonionic detergent is dependent on the type and amount of template-primer. The greatest stimulation is obtained when high concentrations of (dT)12-18-(rA)n or activated salmon sperm DNA are used as template-primers. Little stimulation is obtained with viral 70S RNA or with (dT)12-18- (dA)n. The detergent stimulation appears to be specific for viral reverse transcriptase since this effect is not observed with purified bacterial DNA polymerase or with three known mammalian cellular DNA polymerases. This finding may, therefore, be a useful additional criterion for distinguishing viral reverse transcriptase isolated from cells from other cellular DNA polymerases. Nonionic detergent also has a stabilizing effect on viral DNA polymerase against thermal inactivation. This stabilizing effect is further enhanced by the presence of template-primer.
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PMID:On the stimulation of viral DNA polymerase activity by nonionic detergent. 4 56

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.
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PMID:Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. 4 24

DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.
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PMID:Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. 4 25

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.
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PMID:Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation. 4 26

The RNA-directed DNA polymerase of Rous sarcoma virus requires a 4S RNA molecule as primer for the initiation of DNA synthesis on the viral 70S RNA genome. We have now functionally identified primer activity in uninfected cells on the basis of the capacity of cellular 4S RNA to actively participate in the initiation of DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus in vitro. This was accomplished by reconstitution experiments in which 4S RNA from uninfected avian cells was tested for its ability to restore template activity to the viral RNA genome from which all primer had been removed. Similar reconstitution experiments were employed to demonstrate a primer activity in the 4S RNA population of duck, mouse, and human cells. Primer activity appears to be absent in lower eukaryotic or prokaryotic cells. Unambiguous identification of the Rous sarcoma virus primer molecule in uninfected cells was accomplished by directly purifying a 4S RNA molecule from the bulk of host cell transfer RNA and establishing structural similarities between this cellular 4S RNA species and the Rous sarcoma virus primer by two-dimensional paper electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the RNA species. We conclude that the Rous sarcoma virus DNA polymerase can utilize a host cell molecule as primer for the initiation of RNA-directed DNA synthesis in vitro.
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PMID:RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells. 4 51

Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.
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PMID:RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients. 4 90

Inhibition of DNA polymerase from oncorna viruses by a new class of macromolecular inhibitors is reported. The macromolecule, designated as mercaptopolycytidylic acid (MPC), is a chemically modified polycytidylic acid containing 5-SH cytidylic bases in the polymerase. Partially thiolated polycytidylic acids (MPC I-III, containing 1.7%, 3.5%, and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA-polymerase of Friend leukemia virus (FVL) in the endogenic reaction as well as in the presence of poly rA-(dT)14 or poly (dA-dT) templates; the inhibitory activities were directly related to the percent of tholation. In a bacterial DNA polymerase (E coli-K12 with denatured calf thymus DNA as template) MPCI-III showed no activity. Biological experiments showed that MPC III inhibits the leukemogenic potential of cell-free spleen extracts from FVL-infected mice to about 60%, measured on the basis of spleen weight. The enzymatic and animal experiments have led us to carry out preliminary clinical trials in some cases of Children leukemia. These cases, resistent to the known therapeutic regimes (combination chemotherapy), responded well when treated with MPC along, or in combination with poly I. The experiments indicate that the development of modified polynucleotids with structural similarities to functional templates may be of potential use in the future chemotherapy of leukemia.
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PMID:[Inhibition of viral reverse transcriptase and leukemogenesis by modified nucleic acids (author's transl)]. 4 86


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