Radiolysis of spin-labeled DNA: an electron spin resonance investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.     

Transcriptional regulation of nuclear orphan receptor,NOR-1, by Ca(2+)/calmodulin-dependent protein kinase cascade

It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.     

Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector.

We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E. coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites. Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations. 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction. In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide. At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP. At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP. The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself. In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide.     

The Role of Hydroxyl Radicals in the Degradation of DNA By Ozone

The degradation of the nucleotides dAMP, dGMP, dCMP and dTMP and of calf thymus DNA by ozone was studied. In all cases both base and sugar moiety were degraded. Furthermore, strand breaks were induced in calf thymus DNA. Hydroxyl radicals were probably involved in the oxidation of the base in dAMP and of the deoxyribose ring, but not in the degradation of the other bases. This indicates that ozone-induced DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals.     

The Role of Hydroxyl Radicals in the Degradation of DNA By Ozone

The degradation of the nucleotides dAMP, dGMP, dCMP and dTMP and of calf thymus DNA by ozone was studied. In all cases both base and sugar moiety were degraded. Furthermore, strand breaks were induced in calf thymus DNA. Hydroxyl radicals were probably involved in the oxidation of the base in dAMP and of the deoxyribose ring, but not in the degradation of the other bases. This indicates that ozone-induced DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals.     

The interaction of methylene blue,azure B,and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems

The interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG-dC)]₂, [poly(dA-dT)]₂, and the constituent mononucleotides 2′-deoxyguanosine-5′-monophosphate(dGMP), 2′-deoxyadenosine-5′-monophosphate(dAMP), 2′-deoxycytidine-5′-monophosphate(dCMP), and thymidine-5′-monophosphate(dTMP) have been studied by steady-state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG-dC)]₂ and [poly(dA-dT)]₂ were of the same order of magnitude, so that previously reported (G-C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA-dT)]₂ with increasing ionic strength, while with [poly(dG-dC)]₂ this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π-π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H-bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.     

Miscoding events during DNA synthesis past the nitration-damaged base 8-nitroguanine

8-Nitro-2'-deoxyguanosine (8-NO(2)-dG) DNA adducts are induced by the reactive nitrogen species and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO(2)-dG adduct, an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases (pol). Primer extension reactions catalyzed by pol alpha or beta were strongly retarded at the 8-NO(2)-dG lesion; a fraction of primers was extended past the lesion by incorporating preferentially dCMP, the correct base, opposite the lesion, accompanied by lesser amounts of dAMP and dGMP incorporation. In contrast, primer extension reactions catalyzed by pol eta or a truncated form of pol kappa (pol kappaDeltaC) readily extended past the 8-NO(2)-dG lesion. Pol eta and kappaDeltaC showed more broad miscoding spectra; direct incorporations of dCMP and dAMP were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies induced by pol eta and kappaDeltaC were at least 8 times higher than that of pol alpha or beta. Miscoding frequency and specificity of 8-NO(2)-dG varied depending on the DNA polymerases used. These observations were supported by steady-state kinetic studies. 8-NO(2)-dG adduct may play an important role in initiating inflammation driven carcinogenesis.     

Study of the interaction of triethylenethiophosphamide with nucleotides by mass spectrometry with ionization by fission fragments of californium-252]

Study of interaction of the antitumor alkylating drug triethylenethiophosphoramide (thioTEPA) with nucleotides (dGMP and dCMP) suggests highly perspective employment of 252-Cf fission fragment induced desorption mass spectrometry (252-Cf PDMS) in biochemical pharmacology. Using the 252-Cf PDMS the molecular masses of the unstable, unvolatile, high-molecular substances of biological origin and the chemical adducts or complexes with drugs can be used to establish some structural-functional parameters of the above mentioned biomolecules and their derivatives in microvolumes of the incubation medium. The resulting data may be used for modelling chemotherapeutic processes of "drug-biomolecule-target" type. Using 252-Cf PDMS the complexes (dGMP (thioTEPA) n), n = 1, 2, 3 and (dCMP (thioTEPA) n), n = 1, were obtained. Some quantitative parameters and stability of these complexes were studied. Binding of dGMP with drug in the presence of dCMP was shown preferential. The data are compatible with the predictions concerning the mechanism of the antitumor property of the thioTEPA which can be manifested in the impairment structure of DNA of the malignant cells.     

Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase.

DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.     

The dCMP transferase activity of yeast Rev1 is biologically relevant during the bypass of endogenously generated AP sites

The bypass of AP sites in yeast requires the Rev1 protein in addition to the Pol ζ translesion synthesis DNA polymerase. Although Rev1 was originally characterized biochemically as a dCMP transferase during AP-site bypass, the relevance of this activity in vivo is unclear. The current study uses highly sensitive frameshift- and nonsense-reversion assays to monitor the bypass of AP sites created when uracil is excised from chromosomal DNA. In the frameshift-reversion assay, an unselected base substitution frequently accompanies the selected mutation, allowing the relative incorporation of each of the four dNMPs opposite endogenously created AP sites to be inferred. Results with this assay suggest that dCMP is the most frequent dNMP inserted opposite uracil-derived AP sites and demonstrate that dCMP insertion absolutely requires the catalytic activity of Rev1. In the complementary nonsense-reversion assay, dCMP insertion likewise depended on the dCMP transferase activity of Rev1. Because dAMP insertion opposite uracil-derived AP sites does not revert the nonsense allele and hence could not be detected, it also was possible to detect low levels of dGMP or dTMP insertion upon loss of Rev1 catalytic activity. These results demonstrate that the catalytic activity of Rev1 is biologically relevant and is required specifically for dCMP insertion during the bypass of endogenous AP sites.     

Manganese substantially alters the dynamics of translesion DNA synthesis

The effect of metal ion substitution on the dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase was quantitatively evaluated through steady-state and transient kinetic techniques. Substitution of Mn(2+) for Mg(2+) enhances the steady-state rate of dNMP misinsertion opposite an abasic site by 11-34-fold. At the molecular level, the enhancement in translesion DNA synthesis reflects a substantial increase in the rate of the conformational change preceding phosphoryl transfer for all dNTPs that were tested. This is best illustrated by the biphasic pre-steady-state time course of dAMP insertion opposite an abasic site which indicates that a step after chemistry is rate-limiting for steady-state enzyme turnover. Furthermore, the k(pol) value of 40 s(-1) measured under single-turnover reaction conditions is 20-fold greater than the k(cat) value of 2 s(-1) measured for steady-state enzyme turnover. Finally, the low elemental effect ( approximately 2.4-fold reduction in k(pol)) measured by substituting the alpha-thiotriphosphate analogue for dATP further argues that chemistry is not rate-limiting. In contrast to the biphasic insertion of dAMP, pre-steady-state time courses for the insertion of dCMP, dGMP, or dTMP opposite an abasic site were linear. Nearly identical k(pol) values ( approximately 1 s(-1)) were measured for the insertion of dCMP, dGMP, and dTMP opposite the abasic site using single-turnover conditions. However, the large elemental effects of 27 and 70 measured by substituting the alpha-thiotriphosphate analogues for dCTP and dGTP, respectively, suggest that phosphoryl transfer may be the rate-limiting step for their insertion opposite the abasic site. Various models are discussed in an attempt to explain the effect of metal ion substitution on the dynamics of translesion DNA replication.     

Oxidation of thymine to 5-formyluracil in DNA promotes misincorporation of dGMP and subsequent elongation of a mismatched primer terminus by DNA polymerase

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.     

An unusually high number of direct repeats detected by sequence analysis of the dispersed EcoRI-family fragments in Lupinus luteus L.

The Lupinus luteus genome contains a highly repetitive fraction of sequences named the EcoRI family. Two EcoRI molecules, 1071 and 1079 base pairs in length, were cloned, sequenced and compared. Analysis of the internal-sequence organization revealed a number of short direct repeats. Their involvement in the formation of the EcoRI-family fragments is postulated. Evidence is presented for the dispersed type of genomic organization of the EcoRI-family fragments.Abbreviations AluI, BspRI, EcoRI, Mbo, PstI restriction nucleases - bp base pair - G, A, T, C deoxynucleotides: dGMP, dAMP, dTMP and dCMP - pBR322 and pUC18 plasmids used as cloning vehicles     

Synthesis and biological activity of water-soluble maleimide derivatives of the anticancer drug carboplatin designed as albumin-binding prodrugs

Four platinum (II) complexes (13-16) were synthesized by reacting either [Pt trans-DACH](NO(3))(2) with a 6-maleimidocaproic acid, a 15-maleimido-4,7,10,13-tetroxapentadecanoic acid, and a 6-maleimido-4-oxacaproic ester derivative of cyclobutane-1,1-dicarboxylic acid (CDBA) or [Pt(NH(3))(2)](NO(3))(2) with a 6-maleimido-4-oxacaproic ester derivative of CBDA. Both complexes containing the 6-maleimido-4-oxacaproic ester (15, 16) showed good water solubility (>/=8 mg/mL) and CE experiments revealed rapid binding to human serum albumin and the formation of biadducts with dGMP and dAMP. In the MaTu xenograft model in nude mice, both complexes showed an improved antitumor effect at their maximum tolerated dose (2 x 50 mg/kg carboplatin equivalents) compared to therapy with carboplatin at equimolar dose or at its optimal dose (2 x 75 mg/kg).     

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.     

"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.