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)

Human fibroblast cells treated with benzo[alpha]pyrene (BP), aflatoxin B1 (AFB1) or N-acetoxy-2-fluorenylacetamide (A-AAF) inhibited Snyder-Theilen feline sarcoma virus (ST-FeSV) focus formation. Inhibition of focus formation resulting from chemical treatment was not related to cytotoxic concentrations of chemicals in that little or not effect on cells surviving treatment was observed. Maximum inhibition of focus formation occurred with BP when the cells were treated before infection. By contrast, maximum inhibition of focus formation occurred with A-AAF and AFB1 when the cells were treated after virus infection. Inhibition of focus formation by BP and AFB1 was eliminated when virus infected cells were treated 48-96 h post-infection. While no infectious virus was detected in either chemical treated or untreated ST-FeSV virus infected cultures, comparable levels of virus-directed RNA dependent DNA polymerase enzyme assay (RDDP) activity were found in both treated and untreated cultures. The data show that the inhibitory effect on focus formation is chemically mediated while the inhibition of virus synthesis is not.
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PMID:Feline sarcoma virus in vitro infection of human cells. Influence of chemical carcinogens on focus formation. 8 Nov 13

N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature. The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct. The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I. All polymerases appear blocked at acetylaminofluorene lesions. Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation. T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily. The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts.
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PMID:Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA. 170 68

Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DNA repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coli. The repaired DNA is even efficiently replicated, the egg being "activated" by the microinjection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase alpha. However, the repair appears to be very limited, since it does not restore the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and/or to a very low level of DNA polymerase beta. This system was used to study the effect of diadenosine tetraphosphate (Ap4A) on DNA repair. Ap4A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap4A could be involved in switching off the replication machinery when DNA is badly damaged, thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.
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PMID:Repair of acetyl-aminofluorene modified pBR322 DNA in Xenopus laevis oocytes and eggs; effect of diadenosine tetraphosphate. 211 15

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.
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PMID:The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene. 253 61

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.
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PMID:The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases. 634 28

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase alpha from both calf thymus and human lymphoma cells and DNA polymerase beta from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG lesions when Mg2+ is the divalent cation. Substitution of Mn2+ for Mg2+ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase alpha inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase beta is specific, inserting mainly C even in the presence of Mn2+. The Km for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn2+ is about 0.5 mM. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg2+ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn2+. dNTP alpha S and recA protein increase only the insertion of C. We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.
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PMID:A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts. 649 59

The 3'----5' exonuclease activity of bacteriophage T4 DNA polymerase is found to be blocked in the vicinity of the N-2-acetylaminofluorene (-AAF) adducts to DNA. This observation allowed us to determine the binding spectrum of the -AAF adducts along a given DNA sequence. The mutation spectrum in a forward mutation assay within this same sequence has been established. Comparison between the -AAF binding spectrum and the mutation spectrum shows that there is no direct correlation.
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PMID:DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum. 674 82

Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3H]DNA) of bacteriophage T7. This protein has been partially purified from a 1 M NaCl extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With AAF-[3H]DNA as the substrate, the binding reaction reached equlibrium within 1 hr at 4 degrees C, and the extent of binding ws proportional to the amount of protein added. Complex formation was dependent on both pH and salt concentration and was unaffected by the presence of sulfhydryl-blocking agents. The purest protein fraction also recognizes DNA modified with methylmethane-sulfonate or methylnitrosourea. It shows little or no recognition of single-stranded DNA, double-stranded DNA, supercoiled bacteriophage phiX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4DNA, or UV-irradiated DNA. No endo- or exonuclease activity, DNA polymerase activity, or glucosylase activity for AAF-DNA was detectable in the preparation.
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PMID:Recognition of chemical carcinogen-modified DNA by a DNA-binding protein. 693 15

N-(Deoxyguanosin-C8-yl)-2-(acetylamino)fluorene (dG-C8-AAF) and N-(deoxyguanosin-C8-yl)-2-aminofluorene (dG-C8-AF) are the two major DNA adducts induced by the chemical carcinogen 2-(acetylamino)fluorene (AAF). Molecular modeling shows that, in the DNA double helix, dG-C8-AF can maintain an anti-structure and normal base pairing, while dG-C8-AAF favors a syn-structure and causes base displacement. In the phi X174 RF DNA-Escherichia coli transfection system, it has been found that dG-C8-AF is 7-10-fold less lethal than dG-C8-AAF; these results suggest that these two kinds of DNA adducts may have different effects on DNA replication and that they may be repaired by different pathways. We have investigated the effects of these two kinds of adducts on DNA polymerase III holoenzyme (pol III-H) and DNA polymerase I Klenow fragment (pol I-Kf) mediated DNA synthesis by using carcinogen-modified M13 single-stranded DNA hybridized with 32P-labeled primer as templates. We have found that pol III-H and pol I-Kf replicate through dG-C8-AF with 92% and 62% frequency, respectively; in contrast, these two enzymes replicate through dG-C8-AAF with only 38% and 25% frequency, respectively. AF-adducted DNA shows a more profound sequence specificity in blocking DNA synthesis than AAF-adducted DNA, and the sequence specificities in blocking DNA synthesis for both kinds of adducts differ for pol III-H and pol I-Kf.
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PMID:Effect of aminofluorene and (acetylamino)fluorene adducts on the DNA replication mediated by Escherichia coli polymerases I (Klenow fragment) and III. 770 49

The strong rat liver carcinogen, N-2-acetylaminofluorene, forms mainly two types of guanine adducts at the C-8 position, the acetylaminofluorene adduct (dGuo-C8-AAF) and the aminofluorene adduct (dGuo-C8-AF). We have constructed different oligonucleotides bearing a single AF lesion at each of the guanine residues of the NarI mutagenesis hot spot (G1G2CG3CC) and analysed the structural distortion induced by this DNA adduct according to the sequence context. At position G1 and G2, the deformation induced by the AF adduct is smaller than the deformation induced by the corresponding acetylated form of this adduct (i.e. the AAF adduct at the G1 and G2), whereas both AF and AAF adducts induce a similar structural change when bound to G3. Single-stranded oligonucleotides modified with AF adducts were used in primer extension replication assays using purified DNA polymerases (PolIII holoenzyme, Klenow fragment (exo+ and exo-), Sequenase 2.0) and the data compared to the AAF containing substrates. Translesion synthesis (complete bypass) is found with all tested polymerases when AF adducts are bound to G1 or G2 while little or no bypass is seen when the AF adduct is bound to G3. On the other hand, irrespective of its position within the NarI sequence, AAF adducts completely block DNA synthesis. The results described in this paper show that the sole knowledge of the chemical structure of an adduct neither determines uniquely the conformational change it induces at the DNA level nor its replication properties. Indeed, although AF adducts are in most cases non-distorting adducts and as a consequence non-replication blocking lesions (as exemplified by adducts at G1 or G2), some AF adducts (as at position G3) behave almost as AAF adducts in terms of the structural distortion induced and its replication blocking property. These findings stress the strong modulation by the local sequence context of the structural and biological consequences of a given adduct.
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PMID:N-2-aminofluorene and N-2 acetylaminofluorene adducts: the local sequence context of an adduct and its chemical structure determine its replication properties. 779 Dec 16


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