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)

Poly A+ RNA, isolated from a single 210 day fetal bovine nuchal ligament, was used to synthesize cDNA by the RNase H method, using AMV reverse transcriptase for first strand synthesis and DNA polymerase I for the second strand. The cDNA was inserted into lambda gt10 using EcoRI linkers, and recombinant phage containing elastin sequences were identified by hybridization with a 1.3 kb sheep elastin cDNA clone, pcSELI (Yoon, K. et al., Biochem. Biophys. Res. Comm. 118: 261-265, 1984). Three clones containing the largest inserts of 2.9, 2.8, and 2.6 kb were selected for further study. The complete sequence analysis of the 3 clones was correlated with the sequence of 10.2 kb of the bovine elastin gene. The analyses: (i) showed that the cDNA encompassed the great majority of the translated sequence, (ii) ordered the tryptic peptides of porcine tropoelastin, (iii) determined new amino acid sequences not previously found in the porcine peptides and (iv) demonstrated that alternative splicing of the primary transcript leads to significant variation in the sequence of the translated portion of the mRNA.
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PMID:Sequence variation of bovine elastin mRNA due to alternative splicing. 366 2

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.
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PMID:Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs. 380 92

A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.
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PMID:Isolation and characterization of a DNA primase from human mitochondria. 404 69

Several natural RNAs were compared with respect to their template activities for the DNA polymerase of Rous Sarcoma Virus during a 2-hr incubation period. 60-70S viral RNA was found to be a 5- to 10-fold better template than heat-dissociated Rous viral RNA, influenza virus RNA, tobacco mosaic virus RNA, or ribosomal RNA. Denatured salmon DNA is a little better, and poly(dAT) is 2-4 times better as a template for the enzyme than is 60-70S Rous viral RNA. The 60-70S RNAs of different strains of avian tumor viruses have very similar template activities for a given avian tumor virus DNA polymerase. Oligo(dT) or oligo(dC) were found to enhance the template activity of heat-dissociated Rous viral RNA 20- to 30-fold, and that of other natural RNAs tested one- to several-fold. DNA syntheses of 1-24% were obtained during a 2-hour incubation of the enzyme with the above RNA templates. The results suggest that the enzyme prefers partially doublestranded or hybrid regions of RNAs for optimal DNA synthesis, but certain regions of single-stranded RNA can also serve as templates.Poly(dAT) competes with viral RNA for purified DNA polymerase during DNA synthesis, as would be expected if RNA- and DNA-dependent DNA synthesis was performed by at least one common active site of the same enzyme.
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PMID:Comparative properties of RNA and DNA templates for the DNA polymerase of Rous sarcoma virus. 433 12

DNA polymerase from Micrococcus luteus and RNA polymerase from E. coli catalyze the synthesis of poly(dA) with poly(dT) template, in the presence of ATP and [alpha-32P]dATP. The reaction is completely dependent on poly(A) primer synthesis. Poly(A) chains are covalently extended by DNA polymerase. Primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA). The length of RNA and DNA products appears to be relatively variable. The size of the DNA is less than 3 000 nucleotides.
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PMID:Ribonuclease H from chick embryos cleaves precisely at the junction between the RNA and DNA portion of the hybrid helix. 618 57

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).
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PMID:Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity. 620 83

Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions. Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a [3H]cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.
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PMID:Cloning and analysis of a cDNA coding for bovine prothrombin. 625 59

In vitro poly(dA) synthesis on poly(dT) template can be initiated by poly(A) primer. Poly(A) chains are covalently extended by DNA polymerase. The reaction product consists of poly(dA) chain with poly(A) at their 5'-ends, hydrogen bonded to the template poly(dT). The primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA). Poly- or oligoriboadenylate longer than the (pA)5 could serve as a priming activity to synthesize poly(A) covalently linked to poly(dA).
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PMID:Effect of ribonuclease H from chick embryo on the covalent-linked poly(A)--poly(dA) complementary to poly(dT) template. 629 71

Poly(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli X1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver. 631 66

Poly(dC,3- MedC ) has been synthesised and used as a template to compare the miscoding properties of 3-methylcytosine (3-MeC) during DNA and RNA synthesis. Although 3-MeC was promutagenic with the RNA polymerase incorporating both AMP and UMP in the ratio of approximately 5:1 (agreeing with results reported by earlier workers) no non-complementary nucleotide incorporation was observed with DNA polymerase I. The results show that 3-MeC, which is a strong inhibitor of DNA synthesis, is only promutagenic with the less accurate RNA polymerase and that the reported differences in promutagenicity for this modified base with the two nucleotide polymerising enzymes arise from different specificities for the two enzymes.
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PMID:Differences in the promutagenic nature of 3-methylcytosine as revealed by DNA and RNA polymerising enzymes. 637 42


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