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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)

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.
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PMID:[DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site]. 396 8

In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers. RNA chains are covalently extended by DNA polymerase II from KB cells and DNA polymerase I from Micrococcus luteus, but not by an RNA-dependent DNA polymerase from avian myeloblastosis virus. The reaction product consists of DNA chains with a piece of RNA at their 5'-ends, hydrogen bonded to the template DNA. The primer RNA is linked to the product DNA via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H. The possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed.
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PMID:RNA-primed DNA synthesis in vitro. 433 98

A complex of ribosomal DNA with RNA has been isolated from ovaries of metamorphosing tadpoles of Xenopus laevis. The complex is disrupted by treatments that destroy hydrogen bonds, and the sedimentation of the DNA within the complex is sensitive to RNase. We suggest that the RNA-ribosomal DNA complex is an intermediate in the synthesis of amplified ribosomal DNA and that the RNA is a template. In addition, in a preliminary attempt to mimic the amplification process in vitro, we have demonstrated the use of this RNA from the complex as a template for DNA synthesis by a DNA polymerase isolated from ovaries of Xenopus.
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PMID:[On the role of RNA in gene amplification]. 450 50

Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J/m2, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with Micrococcus luteus correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf thymus DNA polymerase alpha but was recognized as a template by DNA polymerase beta. The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules od dNMP) per one correndonuclease incision. The length of the DNA polymerase beta product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg(2)=nd four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate. The average numbers of deoxynucleotides incorporated per one DNAase I incision or per one nonspecific break, measured in control samples, were equal, amounting to 0.3 dTMP molecule. This value corresponded to 1.2 dNMP molecule; in our opinion, this reflects contaminating nuclease activity of the system used. The present results testify to the ability of DNA polymerase beta to repair synthesis by the "patch and cut' mechanism.
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PMID:The DNA polymerase beta reaction with ultraviolet-irradiated DNA incised by correndonuclease. 625 Jun 19

The covalent closing of hydrogen-bonded lambda DNA circles in Escherichia coli extract was observed to require DNA polymerase I, recBC enzyme and ATP. This covalent closing activity was lost in strains harbouring a mutation in one of the genes responsible for production of the enzymes mentioned above, and was recovered by combining these mutant extracts. ATP could be replaced with dATP, but not appreciably with any of the other nucleoside triphosphates. High concentrations of ATP inhibited the closure. K+ or NH4+ (0.2M) was required for optimal activity and NMN was a strong inhibitor.
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PMID:DNA polymerase I and recBC enzyme support the covalent closing of hydrogen-bonded lambda DNA circles in extracts of Escherichia coli cells. 625 65

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

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.
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PMID:Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. 630 35

O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.
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PMID:Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis. 634 76

Sites on an fd DNA template which terminate synthesis catalyzed by each of four forms of Escherichia coli DNA polymerase III have been identified at single nucleotide resolution. Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3'-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis. Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination. The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing. Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local hydrogen-bonding interactions. The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme. The occurrence of termination sites is markedly affected by the inclusion of spermidine or Escherichia coli single-stranded DNA binding protein in the reaction mixtures. Additionally, a nucleotide composition specificity of pause sites has been observed.
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PMID:Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. 636 Feb 4

N4- Methoxydeoxycytidine triphosphate ( mo4dCTP ) substitutes for dTTP in poly d[A-T] synthesis with E. coli DNA polymerase I (Pol I). In parallel experiments using as template-primer, poly d[G-C], no incorporation of [14C] mo4dC was detected. This indicates that this deoxy derivative acts as the imino tautomer, as previously found for the riboderivative . Nearest neighbor analysis of transcripts of poly d[A-T] containing mo4dC shows that the derivative substitutes for only one base. In replication, singlestranded mo4dC -containing polymers gave little misincorporation, including that of dATP which can hydrogen-bond to mo4dC in the imino form, if the methoxy group is anti to the N-3. It is therefore assumed that the methoxy group is constrained anti in a polymer such as d[A-T], but can be in the syn form in singlestranded polymers and not recognized by DNA polymerase. mo4dC destabilizes the poly d[A-T] helix, as indicated by a lowered and less cooperative melting. Steric factors such as adjacent base displacement were invoked for similar findings with the doublestranded r( U61 , mo4C39 ) X r(A).
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PMID:N4-Methoxydeoxycytidine triphosphate is in the imino tautomeric form and substitutes for deoxythymidine triphosphate in primed poly d[A-T] synthesis with E. coli DNA polymerase I. 637 35


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