The Impact of an Exocyclic Cytosine Adduct on DNA Duplex Properties:  Significant Thermodynamic Consequences Despite Modest Lesion-Induced Structural Alterations

Abstract

The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

Impact of the Oxidized Guanine Lesion Spiroiminodihydantoin on the Conformation and Thermodynamic Stability of a 15-mer DNA Duplex

“…Studies are currently under way in our laboratory to examine the effect of both DNA sequence context and base pairing partner on the thermodynamic impact of the Sp lesion. The most destabilized lesion in this survey, on the basis of ∆∆G 37 values, is 3,N 4 -deoxyethenocytosine, which can arise from exposure to vinyl chloride, hepatic copper poisoning, or reaction of DNA with products of lipid peroxidation (49). This adduct very dramatically reduces the thermodynamic stability of the duplex with a ∆∆G 37 of 15 kcal/mol, representing a ∼85% loss in the total duplex thermodynamic stability (49).…”

The Main Role of Human Thymine-DNA Glycosylase Is Removal of Thymine Produced by Deamination of 5-Methylcytosine and Not Removal of Ethenocytosine

“…One melting temperature study found that on average a G ⅐T mismatch contributes 3.5 kcal/mol less energy than a G ⅐C base pair to the stability of a DNA duplex (37). In another study, 13 bp DNA duplexes containing a G ⅐ ⑀ C base pair were 13.4 -15.3 kcal/mol less stable than the parental G ⅐C-containing duplexes (38). A further study directly comparing the melting temperature of 15-bp DNA duplexes containing either a G ⅐ ⑀ C base pair or a G ⅐T mismatch found that the G ⅐ ⑀ C containing duplexes were 0.43-1.63 kcal/mol less stable than the corresponding G ⅐T-containing duplex (39).…”

Differential Destabilization of the DNA Oligonucleotide Double Helix by a T·G Mismatch, 3,N4-Ethenocytosine, 3,N4-Ethanocytosine, or an 8-(Hydroxymethyl)-3,N4-ethenocytosine Adduct Incorporated into the Same Sequence Contexts

“…Experimental results showed that the T‚G and C‚G mismatches apparently do not either have similar geometry or induce similar structural changes (4)(5)(6)(7)(8). Therefore, the observation that the same enzyme recognizes two structurally dissimilar substrates (2,3) raises the following question (11): What is the common motif in substrate recognition and selection by the human G/T(U) mismatch-DNA glycosylase?…”