O<sup>4</sup>-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T<sub>4</sub>DNA polymerases

Singer, Brett C.

doi:10.1093/nar/14.16.6735

Cited by 6 publications

(2 citation statements)

References 22 publications

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“…The relative ease with which the m4G-T mispair can be extended suggests that it is not a good substrate for proofreading by the "weak" 3'-exonuclease activity of the Klenow fragment. In the absence of any dNTP the exonuclease activity of the Klenow fragment did not excise even m4T-A pairs (Singer, 1986). For the case of another modified base pair, m6G-T, we also found that it was extended much more rapidly to the next template site than the parent G-T base pair (Singer et al, 1989b).…”

Section: Resultsmentioning

confidence: 72%

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Dosanjh¹,

Essigmann²,

Goodman³

et al. 1990

Biochemistry

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Synthesis of a 25-mer oligonucleotide template containing O4-methylthymine (m4T) at a unique site is reported. The sequence used is analogous to that studied previously to determine the mutation frequency of O6-methylguanine in vitro and in vivo. The templates containing m4T or unmodified T were used in a primer-extension gel assay to determine kinetic parameters for incorporation by DNA polymerases of dGTP and dATP opposite either m4T or T. Both Escherichia coli DNA polymerase I (Klenow fragment, Kf) and Drosophila melanogaster polymerase alpha-primase complex (pol alpha) were used. On the basis of the Vmax/Km ratios, the pairing of m4T.G was preferred over that of both m4T.A and T.G by more than 10-fold. The two polymerases gave almost identical values for the frequency of formation of all pairs investigated including m4T.G pairs, suggesting that the 3'----5' exonuclease activity of the Klenow fragment does not efficiently edit such pairs. Extension beyond m4T.G was demonstrated with both Klenow and pol alpha. In similar kinetic experiments, bacteriophage T4 DNA polymerase, which has a very high 3'----5' exonuclease activity, allows stable incorporation of G opposite m4T in contrast to G opposite T. This kinetic approach allows quantitation of the mutagenic potential in the absence of alkylation repair and additionally provides qualitative data on mutagenesis that are in accord with our previous in vivo studies showing that replication of m4T causes T----C transitions.

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Section: Resultsmentioning

confidence: 72%

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Dosanjh¹,

Essigmann²,

Goodman³

et al. 1990

Biochemistry

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“…It is generally assumed that nucleobase analogs can cause replicational errors. Studies with procaryotic polymerases on the incorporation of nucleotide analogs into DNA in vitro have been carried out in many laboratories (12,17,22,26,27,31,34). In contrast, there are only a few reports regarding the utilization of base analogs by eucaryotic DNA polymerases (10,11,33).…”

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confidence: 99%

Induction of Mutation in Mouse FM3A Cells by N⁴-Aminocytidine-Mediated Replicational Errors

Takahashi

Nishizawa

Negishi

et al. 1988

Molecular and Cellular Biology

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To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.

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Proofreading of a mutagenic nucleotide, N4-Aminodeoxycytidylic acid, by DNA polymerase I

Takahashi

Negishi

Hayatsu

1987

Biochemical and Biophysical Research Communications

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O⁴-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T₄DNA polymerases

Cited by 6 publications

References 22 publications

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Induction of Mutation in Mouse FM3A Cells by N⁴-Aminocytidine-Mediated Replicational Errors

Proofreading of a mutagenic nucleotide, N4-Aminodeoxycytidylic acid, by DNA polymerase I

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O4-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T4DNA polymerases

Cited by 6 publications

References 22 publications

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Comparative efficiency of forming m4T.cntdot.G versus m4T.cntdot.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase .alpha.-primase complex

Induction of Mutation in Mouse FM3A Cells by N4-Aminocytidine-Mediated Replicational Errors

Proofreading of a mutagenic nucleotide, N4-Aminodeoxycytidylic acid, by DNA polymerase I

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O⁴-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T₄DNA polymerases

Induction of Mutation in Mouse FM3A Cells by N⁴-Aminocytidine-Mediated Replicational Errors