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M D Topal

Publications and source records attributed to M D Topal.

47 records · Page 3Linked to original sources
PubMed1982

DNA precursors in chemical mutagenesis: a novel application of DNA sequencing.

Recently, we have shown that deoxyribonucleoside residues in the cellular DNA precursor pool are generally more susceptible to methylation than are residues within the DNA duplex. The N-1 position of adenosine, for example, was found to be at least 13,000 times more susceptible to methylation by N-methyl-N-nitrosourea (MNU) than the same site in the DNA. These results suggest that potential sites for alkylation in the double-strand duplex are relatively inaccessible to direct alkylation in vivo. Many of these sites are probably protected from alkylation not only by their position in the interstices of the DNA helix, but also by further in vivo 'packaging' of the DNA in chromatin. We have now used DNA sequencing to demonstrate the incorporation properties of products of the reaction of MNU with dATP and of deoxy-N4-hydroxycytidine triphosphate during DNA replication in vitro by phage T4 DNA polymerase and the 'Klenow' fragment of Escherichia coli pol I. The results suggest that DNA precursor nucleotides due to their greater availability for alkylation, may offer routes for the introduction of alkylated residues into double-stranded DNA.

Alkylation↗
PubMed1982

DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells.

Synchronized C3H/10T1/2 clone 8 cells were treated in vitro with a nontoxic dose of N-methyl-N-nitrosourea during their S phase. Chromatographic isolation of the deoxyribonucleotide DNA precursor pool and measurement of the precursor content per cell showed that a nucleic acid residue in the precursor pool is 190-13,000 times more susceptible to methylation than a residue in the DNA duplex, depending on the site of methylation. This conclusion comes from measurements indicating that, for example, the N-1 position of adenine in dATP is 6.3 times more methylated than the same position in the DNA, even though the adenine content of the pool is only a fraction (0.0005) of the adenine content of the DNA helix. The comparative susceptibility between pool and DNA was found to vary with the site of methylation in the order the N-1 position of adenine greater than phosphate greater than the N-3 position of adenine greater than the O6 position of guanine greater than the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed.

Adenine↗
PubMed1982

DNA in proximity to the site of replication is preferentially alkylated in S phase 10T1/2 cells treated with N-methyl-N-nitroso-urea.

Replicating DNA is more susceptible to modification by N-methyl-N-nitrosourea (MNU), a spontaneously active methylating agent, than bulk DNA. This conclusion is supported by results from two different experimental approaches. First, synchronized C3H 10T1/2 clone 8 cells were treated in S phase with MNU and DNA replicated during the period of treatment was separated from bulk DNA. This was done by digesting the purified DNA with restriction enzymes and retaining the replication fork-associated DNA in nitro-cellulose filters. Second, synchronized C3H 10T1/2 clone 8 cells were exposed to 5-bromodeoxyuridine and [3H]MNU and the density-labelled, replicated DNA was separated in CsCl gradients. Both methods show 2.6 to 5.0 times more [3H]methyl adducts per nucleotide residue associated with replicating DNA than that expected from random methylation. These experiments were done at low MNU concentrations (0.018-0.115 mM) that did not cause any detectable inhibition of DNA synthesis or stimulation of repair replication in 10T1/2 cells.

Animals↗
PubMed1980

Molecular basis for substitution mutations. Effect of primer terminal and template residues on nucleotide selection by phage T4 DNA polymerase in vitro.

The DNA-dependent conversion of incorrect deoxynucleoside triphosphate precursors to monophosphates (turnover) by bacteriophage T4 DNA polymerase was determined using either poly(dA) x (dT) or poly(dG) x (dC) homopolymer templates. Competition between correct and incorrect triphosphates for incorporation into DNA, and the use of chain-terminating dideoxynucleoside triphosphates enabled us to determine the amount of turnover occurring at the end of each strand of the homopolymer duplex (e.g. amount of turnover of dATP occurring at the 3'-OH of poly(dG) and the 3'-OH of poly(dC)). These determinations suggest that nearest neighbor interactions between incoming dNTPs and the growing strand terminal residue play a major role in the occurrence of substitution errors during DNA synthesis in vitro byDNA polymerase. When considered together with existing evidence from studies of turnover (Gillin, F. D., and Nossal, N. G. (1976) J. Biol. Chem. 251, 5225-5232) and direct incorporation (Hall, Z. W., and Lehman, I. R. (1968) J. Mol. Biol. 36, 321-333) these results demonstrate that pyrimidine-pyrimidine and purine-purine as well as purine-pyrimidine oppositions have a role in error production at least during DNA replication in vitro. The implications of these results for the role of the "accessory" replication proteins in maintaining accuracy during the DNA biosynthetic process are discussed.

Base Sequence↗
PubMed1980

Fluorescence of terbium ion-nucleic acid complexes: a sensitive specific probe for unpaired residues in nucleic acids.

The interaction of the lanthanide cation Tb3+ with the phosphate moieties of non-hydrogen-bonded residues of nucleic acids has been shown to result in substantial enhancement of the fluorescence of this cation. The excitation spectrum for this fluorescence is characteristic of the base moiety of the residue to which the Tb3+ is bound, while the emission spectrum is characteristic of the cation itself. The intensity of the fluorescence enhancement, however, is dependent upon the base of the ligand moiety, with G inducing the strongest enhancement, C and T rather less, and A very little. Base-paired residues of nucleic acids induce no such fluorescence enhancement, even though the cation is more tightly bound to double helical regions than to residues in single strands. The enhancement of Tb3+ fluorescence upon binding to non-hydrogen-bonded residues therefore provides a highly specific conformational probe for such residues. This probe has been exploited successfully for the purpose of analyzing the kinetics of reassociation of DNAs (C0t analysis) and as a specific stain for single-strand DNA bands on polyacrylamide gels.

DNA↗
PubMed1976

Complementary base pairing and the origin of substitution mutations.

On the basis of chemical considerations and model building, the Watson-Crick concept of complementary base pairing is extended to a wider range of DNA pairs that A-T and G-C (including A-C, G-T, A-A, G-G and G-A) by invoking imino or enol tautomers (or protonated species) and synisomers. The virtual absence of these additional base pairs from DNA is explained in terms of the low frequency with which these unfavoured forms occur and the two-step mechanism of DNA synthesis, whereby residues are first incorporated by the DNA polymerase and then checked. This base-pairing hypothesis is used to explain the origin, nature and level of spontaneous substitution mutations, their enhancement by base analogues, and the unique effects of certain mutator alleles.

2-Aminopurine↗
PubMed1976

Base pairing and fidelity in codon-anticodon interaction.

Base pairing in codon-anticodon interaction has been investigated in order to understand the basis on which particular base pairs have been selected for or against participation at the wobble position and the basis for codon-anticodon infidelity.

Adenine↗
PubMedDate not supplied

Mechanism of mutagenesis by O6-methylguanine.

O6-methylguanine (O6meG) lesions of double-stranded DNA have been associated with mutation and neoplastic transformation. These lesions can, in principle, be produced by at least three different mechanisms: direct alkylation of G X C base pairs in double-stranded DNA; alkylation of guanine residues in single-stranded regions of DNA associated with replication forks; and alkylation of the DNA precursor pool followed by incorporation of O6-methyl deoxyguanosine triphosphate (O6-medGTP) during DNA replication. DNA biosynthesis subsequent to all three events will generate predominantly O6-meG X T base pairs as O6meG preferentially pairs with T. We show here that O6meG X T base pairs are mutagenic; that transalkylase repair has a direct role in the generation of mutations induced by alkylated pool nucleotides; and that the Escherichia coli mismatch repair system is capable of repairing mutagenic G X T intermediates.

Base Sequence↗