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)

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.
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PMID:Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu. 183 56

A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.
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PMID:Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase. 205 51

By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication.
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PMID:Functional domain for priming activity in the phage phi 29 terminal protein. 234 Oct 40

The sequence selectivity of DNA alkylation by the recently isolated pluramycin antitumour antibiotic DC92-B has been investigated using two methods: a piperidine-induced strand-breaking procedure and a Taq DNA polymerase/linear amplification method. These techniques reveal that guanines are the most reactive sites for alkylation and that the level of adduct formation at these sites is clearly sequence dependent. The highest levels of alkylation occurred at isolated guanines located in 5'-CGT sequences and also at the 5'-G in some 5'-CGG sequences. Isolated guanines in 5'-TGT sequences were also quite reactive. We have also re-examined, in parallel, the sequence selectivity of binding of the structurally-related compound hedamycin: the first known example of a bis(epoxide)-containing, DNA-alkylating pluramycin. Our studies included a more extensive sequence analysis of hedamycin binding than that previously reported and we are able, therefore, to define more precisely the sequence preference. Despite significant differences in the stereochemistry and substitution of their bis(epoxide) sidechains, hedamycin and DC92-B exhibited very similar sequence selectivities in our assays.
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PMID:Comparison of the sequence selectivity of the DNA-alkylating pluramycin antitumour antibiotics DC92-B and hedamycin. 769 48

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3',5')-deoxyadenosine photoproduct. 782 86

Free radicals produce a broad spectrum of damages to DNA, a major proportion of which includes ring fragmentation and contraction products of DNA bases as well as abasic sites. In this study, the mutagenic potential fo two pyrimidine ring fragmentation products, urea and beta-ureidoisobutyric acid (UBA), was analyzed using the i-d region of the Escherichia coli lacI gene contained in a single-stranded f1-K12 phage hybrid vector. Single-stranded DNA was used so that the in vivo interactions between the damage and the DNA polymerase could be assessed in the absence of excision repair. The i-d region contains 20 mutable thymine sites so that 20 separate sequence contexts containing a unique lesion at a position of thymine can be analyzed simultaneously. Urea and UBA residues were uniquely introduced into f1-K12 DNA by chemical and enzymatic methods and primer extension and piperidine analysis of the damage-containing template molecules demonstrated that the potential mutable thymine sites contained randomly distributed lesions. Both fragmentation products were poorly bypassed by DNA polymerases in vitro and in the cell; although in the presence of SOS-induction, UBA was bypassed more efficiently than urea. UBA was a potent premutagenic lesion with a rate of mutation induction more than sixfold above that observed with abasic sites derived from purines. Urea residues were about as mutagenic as abasic sites derived from purines, which in turn were more mutagenic than abasic sites derived from thymine. Mutations derived from urea, UBA and abasic sites were all dependent on SOS-induction of the host cells. Since both urea and UBA were derived from DNA thymine, these data demonstrate that adenine is not routinely inserted opposite products that no longer retain the structural integrity of the pyrimidine ring. Sequence analysis showed that the mutations were targeted at thymine with 62% of the urea-derived mutations being T to C transitions and 62% of the UBA derived mutations being T to A transversions. Thus, the two fragmentation products appeared to direct specific misinsertions. The mutations were not randomly distributed over the i-d region for either fragmentation product and hotspots were observed for both damages. The presence of hotspots suggests that in addition to lesion structure, sequence context plays an important role in base selectivity by DNA polymerases opposite DNA lesions. Energy minimization calculations were used to model the urea and UBA lesions at two contrasting hotspot sites. In both cases, there was significant agreement between the computational and biological data sets.
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PMID:Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis. 810 37

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.
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PMID:The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions. 812 67

Deoxyoligonucleotide 49-mers containing a central cis-syn, trans-syn-I, (6-4), or Dewar photoproduct of TpT were constructed for use in repair and replication studies by ligation of shorter photoproduct-containing oligonucleotides. A (6-4) product-containing 6-mer was prepared in 3.4% yield by 254 nm irradiation of d(AATTAA) and converted in nearly quantitative yield to the Dewar isomer by irradiation with Pyrex- and Mylar-filtered medium-pressure mercury arc light. d(CGAATTAAGC) containing a site-specific cis-syn or trans-syn-I dimer was prepared via automated solid-phase DNA synthesis utilizing photoproduct building blocks. The photoproduct-containing 49-mers were characterized by their susceptibility to base cleavage and a number of enzymes routinely used to map the sites of DNA photoproduct formation. 1 M piperidine at 90 degrees C cleaved the Dewar product faster than the (6-4) product, but did not cleave the cyclobutane dimers. The 3'-->5' exonuclease activity of T4 DNA polymerase was completely blocked by all the lesions except the (6-4) product, which it slowly bypassed. T4 endonuclease V did not cleave the (6-4) or Dewar photoproduct, but unexpectedly cleaved the trans-syn-I dimer at most 1% the rate of the cis-syn dimer in double-stranded DNA. The trans-syn-I dimer was cleaved at a 50-fold higher rate in double- than in single-stranded DNA. Escherichia coli photolyase was found to be specific for the cis-syn dimer at low concentrations. Implications of this work to methodology for mapping and quantifying DNA photoproducts are also discussed.
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PMID:Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl(3'-->5')-thymidine. 849 75

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.
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PMID:Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry. 891 80

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.
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PMID:Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions. 909 64


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