The incorporation of O6-methyldeoxyguanosine monophosphate and O4-methyldeoxythymidine monophosphate into polynucleotide templates leads to errors during subsequent in vitro DNA synthesis

O4-MedTMP and O6-MedGMP may be incorporated from their 5'-triphosphates into polynucleotides by DNA polymerases. Once incorporated O6-MedGMP leads to errors during further cycles of DNA synthesis with a mutagenic efficiency of 0.3-0.7, the value depending upon the relative dNTP concentrations in the assay. In contrast O4-MedTMP leads to very few errors following its incorporation into DNA. The results demonstrate that O6-MedGMP, once incorporated into DNA, should be destined to give rise to DNA base changes during the second round of DNA synthesis.     

The incorporation of O6-methyldeoxyguanosine and O4-methyldeoxythymidine monophosphates into DNA by DNA polymerases I and alpha

The modified nucleoside 5'-triphosphates O6-MedGTP ad O4-MedTTP have been synthesised and their acceptability as DNA-precursors investigated using DNA polymerases I and alpha in an in vitro assay. O6-MedGMP is only incorporated into newly-synthesized DNA-like material in the presence of templates containing thymine bases. Similarly O4-MedTMP is only incorporated in the presence of templates containing guanine bases. The results confirm the promutagenic nature and base-pairing properties of O6-MeG ad O4-MeT.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Kinetics and genetic control of the incorporation of thymidine monophosphate in yeast DNA

Summary Strain 211-1a of the yeast Saccharomyces sp. allows the specific labelling of nuclear and mitochondrial DNA by exogenously supplied radioactive thymidine monophosphate (TMP) during the latter half of log phase growth. Genetic analysis of crosses between derivatives of strain 211-1a and wild-type strains that do not take up TMP showed a 2:2 segregation pattern indicating that a single gene controls the uptake of TMP. The allele allowing the uptake of TMP is recessive in that diploid strains take up TMP only in the homozygous configuration.     

Chemical modification of DNA: quantitation of O6-methyldeoxyguanosine by tandem mass spectrometry.

Methodology is described which allows quantitation of O6-methyldeoxyguanosine generated as a product of in vitro methylation of calf thymus DNA by methyl methanesulfonate (MeMS). Quantitative precision of 10% is achieved on samples of 10(-11)-10(-12) mol generated in 0.02% yield (expressed as O6-methyldeoxyguanosine versus deoxyguanosine) when DNA is treated with the weak carcinogen MeMS. These results show the potential application of this method to the analysis of DNA chemical modifications at the low levels that are relevant to the induction of biological effects of many alkylating agents. The methodology utilizes enzymatic degradation, reverse-phase chromatography and finally analysis by tandem mass spectrometry using desorption chemical ionization. Multiple reaction monitoring was used to increase sensitivity and the CD3-labeled nucleoside was used as an internal standard for quantification.     

Cyclic cytidine monophosphate stimulates DNA synthesis by bovine mammary tissue in vitro

Mammary gland biopsies were taken from midpregnant heifers (n = 4), cut into pieces .5 mm thick and 3 - 5 mm2 and incubated for 48 hours in Eagle's Minimum Essential Medium containing 0, .1 or 1 micrograms/ml insulin and 0, 10(-8), 10(-7), 10(-6), 10(-5), or 10(-4) M dibutyryl cyclic 3', 5', cytidine monophosphate (dbcCMP). With 0 or .1 microgram/ml insulin, dbcCMP decreased incorporation of tritiated thymidine into DNA. Similar declines in DNA synthesis were observed with sodium butyrate, suggesting that the decline was due to the butyrate rather than to a cyclic CMP-specific effect. With 1 micrograms/ml insulin, dbcCMP increased DNA synthesis. Higher levels of dbcCMP reduced DNA synthesis relative to 10(-6)M dbcCMP, as did sodium butyrate. Thus cCMP is capable of stimulating mammary growth.     

Structural studies of O6-methyldeoxyguanosine and related compounds: a promutagenic DNA lesion by methylating carcinogens.

O6-Methylation of guanine residues in DNA can induce mutations by formation of base mispairing due to the deprotonation of N(1). The electronic, geometric and conformational properties of three N(9)-Substituted O6-methylguanine derivatives, O6-methyldeoxyguanosine (O6mdGuo), O6-methylguanosine (O6mGuo) and O6, 9-dimethylguanine (O6mdGua), were investigated by X-ray and/or NMR studies. O6mdGuo crystallizes in the monoclinic space group P2(1) with cell parameters a = 5.267(1), b = 19.109(2), c = 12.330(2) A, beta = 92.45(1) degrees, V = 1239.8(3) A3, z = 4 (two nucleosides per asymmetric unit), and O6mGua in the monoclinic space group P2(1)/n with cell parameters a = 10.729(2), b = 7.640(1) c = 10.216(1) A, beta = 92.17(2) degrees, V = 836.7(2) A3, z = 4. The geometry and conformation of O6-methylguanine moieties observed in both crystals and are very similar. Furthermore, the molecular dimensions of the O6methylguanine residue resemble more closely those of adenine than those of guanine. The methoxy group is coplanar with the purine ring, the methyl group being cis to N(1). The conformation of O6-methylguanine nucleosides is variable. The glycosidic conformation of O6mdGuo is anti for molecule (a) and high-anti for molecule (b) in the crystal, while that of O6mGuo is syn [Parthasarathy, R & Fridey, S. M. (1986) Carcinogenesis 7, 221-227]. The sugar ring pucker of O6mdGuo is C(2')-endo for molecule (a) and C(1')-exo for molecule (b). The C(4')-C(5') exocyclic bond conformation in O6mdGuo is gauche- for molecule (a) but trans for molecule (b), in contrast with gauche+ for O6mGuo. The hydrogen bonds exhibited by O6-methylguanine derivatives differ from those in guanine derivatives; the amino N(2) and ring N(3) and N(7) atoms of O6-methylguanine residues are involved in hydrogen bonding. 1H-NMR data for O6mdGuo and O6mdGuo reveal the predominance of a C(2')-endo type sugar puckering. In O6mdGuo, however, a contribution of a C(1')-exo sugar puckering is significant. The NOE data also indicate that O6mdGuo molecules exist with nearly equal population for anti (including high anti) and syn glycosidic conformations. These observations and their biological implications are discussed.     

Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O⁶ position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O⁶-methyldeoxyguanosine (O⁶-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O⁶-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O⁶-methylguanine (O⁶-meG). Exposure of cells to 0.6 mM O⁶-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O⁶-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O⁶-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O[su6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O⁶-medGuo, it is concluded that this lesion is highly pro-mutagenic.     

Effect of DNA polymerase beta loop variants on discrimination of O6-methyldeoxyguanosine modification present in the nucleotide versus template substrate

We have examined the mechanism of DNA polymerase beta (pol beta) lesion discrimination using alkylated dNTP versus alkylated DNA template substrates and the pol beta variants R253M and E249K. Both of these amino acid variants are located in the loop region of the palm domain and are known to play a role in pol beta fidelity and discrimination of 3'-azido-3'-deoxythymidine triphosphate substrates. We observed that these variants affect O(6)-methyldeoxyguanosine- (m6G-) modified dNTP discrimination without affecting m6G template translesion synthesis. Under steady-state conditions, the ratio of inherent reactivity values for the m6dGTP substrate relative to the dGTP substrate was greater for both variant polymerases than for wild-type (WT) pol beta. Biochemical assays of translesion synthesis using m6G lesion-containing templates demonstrated no significant differences between the variants and WT. Using N-methyl-N-nitrosourea- (MNU-) modified DNA templates in the HSV-tk in vitro assay, no difference among the enzymes in the frequency of alkylation-induced G to A transition mutations was observed. However, differences among the polymerases in the frequency of alkylation-induced C to A transversions were observed, consistent with a mutator tendency for E249K and an antimutator tendency for R253M. We conclude that a specific interaction at the loop of the palm domain is involved in pol beta discrimination of the m6G lesion when present on the incoming dNTP substrate but not when present in the DNA template. Our data support a role for the flexible loop in pol beta error discrimination.     

Differential incorporation of halogenated deoxyuridines during UV-induced DNA repair synthesis in human cells

Double labeling of interphase and metaphase chromosomes by 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) has been used in studies of the dynamics of DNA replication. Here, we have used this approach and confocal microscopy to analyze sites of DNA repair synthesis during nucleotide excision repair (NER) in quiescent human fibroblasts. Surprisingly, we have found that when both precursors are added at the same time to UV-irradiated cells they label different sites in the nucleus. In contrast, even very short periods of simultaneous IdU+CldU labeling of S-phase cells produced mostly overlapped IdU and CldU replication foci. The differential labeling of repair sites might be due to compartmentalization of I-dUTP and Cl-dUTP pools, or to differential utilization of these thymidine analogs by DNA polymerases delta and epsilon (Poldelta and Polepsilon). To explore the latter possibility we used purified mammalian polymerases to find that I-dUTP is efficiently utilized by both Poldelta and Polepsilon. However, we found that the UV-induced incorporation of IdU was more strongly stimulated by treatment of cells with hydroxyurea than was incorporation of CldU. This indicates that there may be distinct IdU and CldU-derived nucleotide pools differentially affected by inhibition of the ribonucleotide reductase pathway of dNTP synthesis and that is consistent with the view that differential incorporation of IdU and CldU during NER may be caused by compartmentalization of IdU- and CldU-derived nucleotide pools.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.     

The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis

The frequency and specificity of mutations produced during in vitro DNA synthesis of the lacZ alpha gene in M13mp2 DNA by eucaryotic DNA polymerase-alpha (pol-alpha) and DNA polymerase-gamma (pol-gamma) have been determined. Pol-alpha, purified from five different sources, produces mutations resulting in loss of alpha-complementation at a frequency of 0.8-1.6%/single round of gap-filling DNA synthesis. DNA sequence analysis of 420 independent mutants produced by pol-alpha demonstrates three classes of errors. The majority of mutations result from single base substitutions, while single base frameshifts are detected at a lower but substantial frequency. Large deletions are also observed, with a frequency and specificity suggesting that they too are produced by pol-alpha in vitro. In contrast, pol-gamma is more accurate, producing mutants at a frequency of 0.3-0.5%. The specificity of pol-gamma errors is also different, since more than 90% of the mutants result from single base substitutions, while frameshift errors are not observed at a frequency significantly above background. The pol-gamma mutant spectrum also contains deletion mutations (10 of 179 mutants) presumably resulting from aberrant in vitro synthesis. When considered together with previous results using pol-beta (Kunkel, T. A. (1985) J. Biol. Chem. 260, 5787-5796) the relative accuracy of the three classes of purified vertebrate DNA polymerases for base substitutions, frameshifts, and deletions is in the order gamma greater than alpha greater than beta. These data demonstrate a correlation between the accuracy and processivity of DNA polymerization. Thus, the most accurate DNA polymerase (pol-gamma) also incorporates the most nucleotides per association with the primer-template, while the least accurate enzyme (pol-beta) is the least processive. This correlation exists both for base substitution mutations and for single base frameshifts, and is most obvious for minus-one-base frameshifts in runs of pyrimidines. In support of this correlation, increasing the processivity of pol-beta from 1 to 4-6 incorporations per association increases the accuracy of in vitro DNA synthesis by severalfold. The data imply that the processivity of DNA synthesis could be an important factor in controlling the levels of spontaneous and perhaps induced mutations.     

Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O(6)-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) delta also copied past these two lesions but was totally blocked by O(6)-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols iota and kappa produced mainly only 1-base incorporation opposite O(6)-MeG and O(6)-BzG and had very low activity in copying O(6)-PobG. Human pol eta copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O(6)-alkylG adducts for dCTP and dTTP with pol eta and kappa; pol iota showed a strong preference for dTTP. pol eta, iota, and kappa showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O(6)-MeG but little, if any, for O(6)-BzG or O(6)-PobG. Analysis of the pol eta O(6)-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol eta primer extension products indicated multiple components, mainly with C or T inserted opposite O(6)-alkylG but with no deletions in the cases of O(6)-MeG and O(6)-PobG. With pol eta and O(6)-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O(6)-BzG). The results with pol eta may be relevant to some mutations previously reported with O(6)-alkylG adducts in mammalian cells.     

In vitro incorporation of tritium into native DNA

An exchange method is described for producing tritium-labeled native DNA in vitro with minimal physical damage to the DNA. Tritium-labeled calf thymus DNA prepared in this way has a specific activity of about 100 μCi/mmole of nucleotide (i.e., about 2 × 10⁸ dpm/mmole). Sedimentation velocity at neutral and alkaline pH indicate that the product has an average of two single strand breaks per duplex molecule of molecular weight 6 × 10⁶ daltons. The optical and thermal denaturation properties of the product are those of native DNA. The method should be particularly useful for labeling DNA from organisms that cannot be labeled conveniently in vivo.