Our laboratory has been studying factors that induce deamination in DNA. We have initiated a series of studies on model nucleic acid base and polymer systems to examine two interrelated hypotheses: (1) that ionized base pairs may be found in DNA, and (2) they may provide one pathway that can lead to induced deamination in DNA. We hypothesize that protonated base pairs may arise from several different kinds of DNA alterations, and that the deamination of protonated cytosines in these bases may comprise a previously undetermined source of genetic mutations. In order to study induced deamination, we have developed a reversion assay by which the rates of deamination can be assessed under a variety of conditions. In the present grant, we propose to extend our studies in several ways. We will test the hypotheses that an aberrant base like O(6)-methylguanine in one strand of DNA will induce deamination of the cytosine in the opposite strand, and that protonated cytosine residues in triple helices may deaminate with accelerated rates as compared to normal B-form DNA. First, we will continue our synthetic and site-directed mutagenesis studies to determine the rate and conditions under which cytosine deaminates in O(6)- alkylguanine:cytosine base pairs in DNA. By varying the nature of the O(6)-alkyl substituent, we can examine the effect of electron-donating and electron withdrawing groups on induced deamination. In this way, we hope to discriminate between two possible mechanisms of proton-induced deamination: i.e., local denaturation vs. trapped proton. Second, we will examine the propensity of cytosine to deaminate when it is bulged or is positioned opposite certain modified bases like 2-aminopurine. Third, we will investigate the effect of DNA secondary structure on deamination, including cytosine in an A-form or Z-form conformation or in a hairpin or cruciform structure,. We will ask whether the deamination is kinetically enhanced in DNA conformations where the cytosine can be protonated, as in triple-stranded helices. Fourth, we will continue NMR and calorimetric studies on model DNA nucleosides with the intent of obtaining accurate enthalpies and free energies of hydrogen bonding interactions. The electronic effects of a series of O(6)-alkyl substituents on these interactions will be evaluated.
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| MacPhail, R A; Williams, L D; Jones, D A et al. (1992) Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization. J Biomol Struct Dyn 9:881-98 |
| Frederico, L A; Kunkel, T A; Shaw, B R (1990) A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy. Biochemistry 29:2532-7 |
