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DNA can be damaged by many compounds in our environment, and the resulting damaged DNA is commonly replicated by translesion synthesis (TLS) polymerases. Because the mechanism and efficiency of TLS are affected by the type of DNA damage, obtaining information for a variety of DNA adducts is critical. However, there is no structural information for the insertion of a dNTP opposite an O6-dG adduct, which is a particularly harmful class of DNA lesions. We used molecular dynamics (MD) simulations to investigate structural and energetic parameters that dictate preferred dNTP insertion opposite O6-benzyl-guanine (Bz-dG) by DNA polymerase IV, a prototypical TLS polymerase. Specifically, MD simulations were completed on all possible ternary insertion complexes and ternary -1 base deletion complexes with different Bz-dG conformations. Our data suggests that the purines are unlikely to be inserted opposite anti- or syn-Bz-dG, and dTTP is unlikely to be inserted opposite syn-Bz-dG, because of changes in the active site conformation, including critical hydrogen-bonding interactions and/or reaction-ready parameters compared to natural dG replication. In contrast, a preserved active site conformation suggests that dCTP can be inserted opposite either anti- or syn-Bz-dG and dTTP can be inserted opposite anti-Bz-dG. This is the first structural explanation for the experimentally observed preferential insertion of dCTP and misincorporation of dTTP opposite Bz-dG. Furthermore, we provide atomic level insight into why Bz-dG replication does not lead to deletion mutations, which is in contrast with the replication outcomes of other adducts. These findings provide a basis for understanding the replication of related O6-dG adducts.

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.

Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson-Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N(4)-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.

We present the mid-IR (400-1800 cm(-1)) spectra of 9-ethyl guanine, guanosine, and 2-deoxyguanosine measured by IR-UV double-resonance spectroscopy. We compare the recorded mid-IR spectra with the spectra of the most stable structures obtained from RI-MP2 and RI-DFT-D calculations. The results confirm the enol form for all structures and demonstrate the efficacy of a new approach to DFT calculations that includes dispersion interactions.

In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) studies of crystals X-irradiated at 10 K detected evidence for three radical forms. Radical R1, characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen alpha-couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling, was identified as the primary electron-loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1' of the ribose moiety. The identification of radicals R1-R3 was supported by density functional theory (DFT) calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of one-electron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty.

The dinucleotide d(pGpG) is an often employed DNA model to study various kinds of interactions between DNA and metal ions, but its acid-base properties were not yet described in detail. In this study the six deprotonation reactions of H4[d(pGpG)]+ are quantified. The acidity constants for the release of the first proton from the terminal P(O)(OH)2 group (pKa = 0.65) and for one of the (N7)H+ sites (pKa = 2.4) are estimated. The acidity constants of the remaining four deprotonation reactions were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO3): The pKa values for the deprotonations of the second (N7)H+, the P(O)2(OH)-, and the two (N1)H sites are 2.98, 6.56, 9.54 and 10.11, respectively. Based on these results we show how to estimate acidity constants for related systems that have not been studied, e.g. pGpG, which is involved in the initiation step of a rotavirus RNA polymerase. The relevance of our results for nucleic acids in general is briefly indicated.

This work presents evidence that photo-excitation of guanine radical cations results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. In dsDNA at low temperatures, formation of C1'* is observed from photo-excitation of G*+ in the 310-480 nm range with no C1'* formation observed > or =520 nm. Illumination of guanine radical cations in 2'dG, 3'-dGMP and 5'-dGMP in aqueous LiCl glasses at 143 K is found to result in remarkably high yields (approximately 85-95%) of sugar radicals, namely C1'*, C3'* and C5'*. The amount of each of the sugar radicals formed varies dramatically with compound structure and temperature of illumination. Radical assignments were confirmed using selective deuteration at C5' or C3' in 2'-dG and at C8 in all the guanine nucleosides/tides. Studies of the effect of temperature, pH, and wavelength of excitation provide important information about the mechanism of formation of these sugar radicals. Time-dependent density functional theory calculations verify that specific excited states in G*+ show considerable hole delocalization into the sugar structure, in accord with our proposed mechanism of action, namely deprotonation from the sugar moiety of the excited molecular radical cation.

The formation of binary Ni(II) complexes with 2'-deoxyguanosine 5'-triphosphate (dGTP, L) as well as ternary complexes thereof with L-histidine (His, A) was studied with the use of potentiometry and electronic absorption spectroscopy. In the binary and ternary systems, the complexes with stoichiometries NiH2L-, NiHL2-, NiL3- and NiH2LA2-, NiHLA3-, NiLA4- respectively, were detected. The ternary complexes are very stable at pH 7.4 and thus may constitute biologically relevant Ni(II) carriers in the cell. In the presence of hydrogen peroxide, the binary and ternary systems both generate hydroxyl radical-like species and undergo dGTP degradation with the formation of the 8-oxo-dGTP intermediate. The latter, along with dGTP complexation and degradation, may lead to mutagenesis and carcinogenesis due to base-mispairing properties of 8-oxoguanine and the disturbance in the physiological balance among the four canonical triphosphodeoxynucleotide substrates for DNA synthesis.

We have discovered that short guanine-rich oligonucleotides are able to self-associate into higher order structures that stimulate DNA synthesis in vitro without the addition of a conventional template [Ying, J., Bradley, R. K., Jones, L. B., Reddy, M. S., Colbert, D. T., Smalley, R. E., and Hardin, S. H. (1999) Biochemistry 38, 16461-16468]. Our initial analysis indicated the importance of the presence of three contiguous guanines (G) in an oligonucleotide that stimulates DNA polymerization. To gain insight into and to refine sequence requirements for the unexpected DNA synthesis, we analyzed a 231-member guanine-rich octamer library in a fluorescent nucleotide polymerization assay. We observe that, in addition to three contiguous Gs, the presence of a secondary G cluster within the octamer is essential. Furthermore, the location of the primary G cluster in the center of the molecule is most stimulatory. The majority of the octamers that form extended DNA products have a single non-G base separating the primary and secondary G clusters, the identity of which is predominantly thymine (T). Further, a T 5' or 3' of the primary G cluster positively influences the stimulatory function of the oligonucleotide. Overall, the occurrence of bases in the octamer is in the descending order of G > T > A > C. Our studies demonstrate that structures stabilized by noncanonical base pairings are recognized by a DNA polymerase in vitro, and these findings may have relevance within the cell. In particular, the features of these G-rich stimulatory sequences show striking similarities to telomeric sequences that form diverse G-quartet structures in vitro.

Cadmium is a potent carcinogen in rodents and has recently been accepted by the International Agency for Research on Cancer as a category 1 (human) carcinogen, but the molecular mechanism of its action remains largely unclear. It has however been suggested that cadmium-induced carcinogenesis may involve either direct or indirect interaction of Cd(2+) with DNA. In this study it is found that when Cd(2+) is allowed to interact with adenine and guanine, there is a marked change in the high performance liquid chromatography (HPLC) retention time for adenine but not for guanine. Since Cd(2+) is believed to bind covalently to adenine and guanine, the changes in retention time but absence of any cadmium in the peak fraction point to the following: (i) lability of cadmium-nucleobase adducts, and (ii) introduction of some kind of chemical modification in adenine but not in guanine as a result of covalent binding. This result is different from that for Ni(2+) in which case a change in retention time was observed for guanine but not for adenine.

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.

6-methylated guanine dinucleotides were used to study the influence of hydrogen bonding on the specific binding of the antitumor drug cDDP, cis-PtCl2(NH3)2, to DNA. In this interaction, the guanine-06 site appears to be important in explaining the preference for a pGpG-N7(1),N7(2) chelate, which results from H-bridge formation with the ammine ligand of cDDP. Guanine-06 methylated dinucleotides and the nonmodified dinucleotides were reacted with [Pt(dien)Cl]+, cis-PtCl2(NH3)2, and cis-[Pt(NH3)2(H2O)2]2+ and the reaction products were characterized by 1H NMR using pH titrations. Methylation at guanine-06 clearly reduces the preference for the guanine. In competition experiments monitored by NMR and experiments using UV spectrophotometry a decreasing reactivity towards [Pt(dien)(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+ was found, in the order of d(GpG) greater than d(GomepG) greater than d(GpGome) greater than d(GomepGome). The difference in reactivity between 5' guanine methylation and 3' guanine methylation is ascribed to differences in the H-bond formation with the backbone phosphate. The resulting reduced stacking of the bases in both modified dinucleotides, compared to the bases in d(GpG), results in a preference for the 3' guanine over 5'.

A 32P-postlabelling method was developed to measure 7-methylguanine in human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-me-dGMP) was isolated from normal nucleotides using strong anion-exchange column chromatography. Overall the method gave 35-45% yield as measured with DNA methylated with tritiated dimethyl sulfate. Total white blood cell DNA from healthy non-smokers (n = 17) contained from 2.5 7-methylguanine residues/10(7) nucleotides, corrected for the losses in preparation. Among four patients sampled immediately after a total dose of 1050-2800 mg of dacarbazine or procarbazine, the mean adduct level was 57 7-methylguanine residues/10(7) nucleotides. As further method development, we also investigated the phosphorylation reaction by T4 polynucleotide kinase using dinucleotides containing 7-methylguanine and corresponding imidazole ring-opened products as substrates. We found that imidazole ring-opened dTpdG-Me is resistant to digestion with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. It is quantitatively phosphorylated at femtomolar levels. This method is shown to be suitable for the detection of 7-methylguanine in DNA, and is suggested to be the approach most suited to postlabelling large and labile 7-alkylguanines in DNA.