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

Abstract

The T.G mismatch and the exocyclic adduct 3,N(4)-ethenocytosine (epsilonC) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or epsilonC base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T.G and epsilonC-containing 15-mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5'-TTXTT, 5'-AAXAA, 5'-CCXCC, or 5'-GGXGG sequences, where X is T, epsilonC, or two closely related structural derivatives of epsilonC: 3,N(4)-ethanocytosine (EC) and 8-(hydroxymethyl)-epsilonC (8-HM-epsilonC). Each of the four lesions, incorporated opposite G, decreased both the thermal (T(m)) and thermodynamic stability (DeltaG degrees (37)) of the 15-mer control duplexes. On the basis of the T(m) and DeltaG degrees (37) values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-epsilonC > EC > epsilonC > T.G. The DeltaT(m) values range from -15.8 to -9.5 degrees C when C(t) = 8 microM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2 degrees C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4 degrees C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3. 0 degrees C. Destabilizations were enthalpic in origin. Thus, this work shows that independently changing the modified base, the sequence, or the base opposite the lesion each affects the stability of the duplex, to significantly varying extents. The potential contribution of the thermodynamic stability to repair efficiency is discussed.

8-(Hydroxymethyl)-3,N4-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

“…Similar planar and sugar conformations of εC and 8-HM-εC can lead to the similar stacking potential of the modified base with the neighboring bases, and the ability to form a stable pair with the incorporated base, thus affecting replication efficiency and specificity. In another study on the thermodynamic stability of 15-mer duplexes with an εC or 8-HM-εC paired with G, both adducts showed similar destabilization of the double helix (35).…”

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

“…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). Interestingly, the difference in stability between G ⅐ ⑀ C and G ⅐T was greatest when the mismatch was in a CpG site.…”

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

“…34 The enthalpically stabilizing/entropically destabilizing effect of the dimer lesion is not a feature generally shared by base lesions. 24,35–37 However, like many other lesions, the enthalpic and entropic effects tend to compensate for each other, leading to an overall modest change in free energy. 38 As demonstrated elegantly by the Breslauer group, structure and energetics need not relate directly, and a modest free change in the free energy of overall duplex formation might correspond to a large structural change or localized disturbances counterbalanced by the plasticity of the DNA structure.…”