Sági J, Perry A, Hang B, Singer B
Donner Laboratory, Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Chem Res Toxicol. 2000 Sep;13(9):839-45. doi: 10.1021/tx000040g.
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.
T·G错配和环外加合物3,N(4)-乙烯基胞嘧啶(εC)由同一种酶——人G/T(U)错配-DNA糖基化酶(TDG)修复。该酶从双链DNA中去除T、U或εC碱基。发现切割速率因损伤而异，并且也受相邻序列的影响[杭，B.，梅迪纳，M.，弗伦克尔-康拉特，H.，和辛格，B.(1998年)美国国家科学院院刊95, 13561 - 13566]。由于序列影响双链稳定性，我们测定了含有T·G和εC的15聚体双链体的热力学稳定性，其中损伤两侧的碱基被系统地改变。双链体包含中心5'-TTXTT、5'-AAXAA、5'-CCXCC或5'-GGXGG序列，其中X是T、εC或εC的两个密切相关的结构衍生物：3,N(4)-乙烯基胞嘧啶(EC)和8-(羟甲基)-εC(8-HM-εC)。与G相对掺入的四种损伤中的每一种都降低了15聚体对照双链体的热稳定性(Tm)和热力学稳定性(ΔG°(37))。根据Tm和ΔG°(37)值，15聚体双链体中TTXTT序列的去稳定化顺序如下：8-HM-εC > EC > εC > T·G。当C(t)=8 μM时，ΔTm值范围为-15.8至-9.5℃。侧翼为AA或TT的双链体比侧翼为GG或CC的双链体更不稳定，平均相差2℃。修饰碱基对面的碱基也影响双链稳定性。在TT环境中，加合物对面的四个变化碱基中，C具有最大的去稳定化作用，高达-18.4℃。相反，加合物对面的G通常是最不容易去稳定化的，最小值为-3.0℃。去稳定化起源于焓变。因此，这项工作表明，独立改变修饰碱基、序列或损伤对面的碱基都会影响双链体的稳定性，程度差异很大。讨论了热力学稳定性对修复效率的潜在贡献。
