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

Abstract

Synchronized C3H/IOTI/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-i 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-i position of adenine > phosphate > the N-3 position of adenine > the 06 position of guanine > the N-7 position of guanine. The significance of these results for chemical mutagenesis and carcinogenesis is discussed.We recently proposed, from chemical considerations, that the pool of DNA precursor deoxyribonucleotides should be a significant target in vivo for chemical mutagens (1). Such chemical modification of the precursor pool could have significant biological consequences. For example, modified precursors might inhibit nucleotide binding enzymes and perturb DNA replication. Indeed, modification of precursors together with rapid turnover of the nucleotide pool (for discussion, see ref.2) may offer a direct explanation for the S-phase dependence of alkylation-induced mutation (3) and neoplastic transformation (4, 5) of cells in culture and the chromosome replication point specificity of alkylating agents in bacteria (6, 7). In support of our proposal, dATP was shown to be a good target in vitro for Nmethyl-N-nitrosourea (MNU), a potent methylating agent, mutagen, and carcinogen (1). Moreover, methylated nucleotide products from this reaction were found to incorporate into DNA during replication by bacteriophage T4 DNA polymerase in vitro (1). For these in vitro results to be applicable in vivo, however, the DNA precursor pool must be shown to be a significant target for MNU.In this paper, we report that the DNA precursor pool in mouse embryo fibroblast C3H/1OT'/2 clone 8 cells (lOT'/2 cells) (8) is modified to a greater extent per cell than is the DNA. Furthermore, determination of the cellular content of deoxyribonucleotide residues in the precursor pool enables us to conclude that the purine nucleotide residues in the pool are 190-13,000 times more susceptible to modification by MNU than the same residues in the DNA helix, depending on the site being modified. The order of susceptibility was found to be the N-1 position of adenine > phosphate > the N-3 position of adenine > the 06 position of guanine > the N-7 position of guanine. MATERIALS AND METHODS Deoxyribonucleotides, Deoxyribonucleosides, and Methylated Purine Standards. Purity of dNTPs and deoxyribonucleosides (Sigma) was det...

DNA precursor metabolism and genomic stability

“…However, the concept of damaged nucleotides as premutagenic agents originated 10 years earlier, when Topal and Baker (92) showed that treating cells with a DNA-methylating agent led to far more methylation of free nucleotides than of nucleotide residues in DNA. Whether those modified nucleotides were subsequently incorporated into DNA in significant amounts has still not been established.…”

Deoxyribonucleotide metabolism, mutagenesis and cancer

“…8-oxoguanine in DNA readily base pairs with adenine, initiating a transversion mutagenesis pathway. The idea that the damage might actually occur at the nucleotide level originated from the demonstration that free intracellular nucleotides are orders of magnitude more reactive with modifying reagents than are the corresponding nucleotides in DNA 111 . The idea gained impetus with the finding that the mutT gene in E. coli, which counteracts ROS-stimulated mutagenesis, encodes a nucleoside triphosphatase, which preferentially cleaves the oxidized nucleotide 8-oxo-dGTP to the corresponding deoxyribonucleoside monophosphate 8-oxo-dGMP plus pyrophosphate 112 .…”

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

“…A primary oxidative base modification in DNA is the oxidized form of guanine, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)567. Furthermore, purine deoxynucleoside triphosphates (dNTPs) in the nucleotide pool are vulnerable to oxidation leading to oxidized forms of DNA precursors, that is, 7,8-dihydro-8′-oxo-dGTP (8-oxodGTP), 7,8-dihydro-8′-oxo-dATP (8-oxodATP) and 2-hydroxy-2′-deoxyadenosine-5′-triphosphate (2-OH-dATP)8910. Oxidized dNTPs can be incorporated into DNA during DNA replication and repair resulting in deleterious effects111213.…”