COMPARISON BETWEEN DNA MELTING THERMODYNAMICS AND DNA-POLYMERASE FIDELITY
COMPARISON BETWEEN DNA MELTING THERMODYNAMICS AND DNA-POLYMERASE FIDELITY
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DOI：
10.1073/pnas.85.17.6252
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发表时间：
1988-09-01
影响因子：
11.1
通讯作者：
TINOCO, I
中科院分区：
文献类型：
--
作者：
PETRUSKA, J;GOODMAN, MF;TINOCO, I
通过使用DNA引物-模板双链体研究DNA聚合酶保真度和碱基配对稳定性之间的关系，所述DNA引物-模板双链体含有共同的9-碱基模板序列，但具有正确的（A. T）或不正确（G. cntdot. T，C. T，T. cntdot. T）引物3“末端的碱基对。热熔融和酶动力学测量进行了比较，为每种终端。解链温度的分析发现，解离时自由能变化之间的差异（Δ.Δ G °）只有0.2、0.3和0.4千卡。mol-1（1 cal = 4.18 J），对于末端A. T与G. cntdot相比。T，C. T和T. T错配，分别在37 ℃。我们表明，焓变与水溶液中DNA中正常和异常碱基对的熵变直接相关，并且Δ。DELTA.G.degree.值很小，因为对应的焓和熵分量几乎抵消。用纯化的果蝇DNA聚合酶α测量引物末端延长的动力学。匹配的A. cntdot.发现T末端延长。比G. cntdot快200倍。T错配，比C. cntdot快1400和2500倍。T和T.cntdot. T错配，分别。针对延长错配末端的酶促区分主要基于Km而不是Vmax差异。由37 ℃时的Km，我们得到Δ。DELTA.G.degree.值为2.6-3.7 kcal. mol-1，约为一个数量级，大于熔融数据所示。核苷酸插入动力学的类似测量先前已经发现形成A. T碱基对比G. cntdot大5000倍。T错配和20，000倍大于C. cntdot。T和T.cntdot. T错配。在此，Km差异也是造成区分的主要原因，并且指示甚至更大的Δ。DELTA.G.degree.值（4.3-4.9 kcal. cntdot.）mol-1）。因此，聚合酶活性位点裂缝中正确和不正确碱基对之间的自由能差异似乎是水性介质中的> 10倍。我们探索的想法，紧密配合正确的碱基对，并排除水在活性位点的结合裂缝可能会放大碱基对自由能差，减少熵差和增加焓差足以占核苷酸插入和延伸保真度。
The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer- template duplexes that contain a common 9-base template sequence but have either correct (A .cntdot. T) or incorrect (G .cntdot. T, C .cntdot. T, T .cntdot. T) base pairs at the primer 3'' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (.DELTA..DELTA.G.degree.) are only 0.2, 0.3, and 0.4 kcal .cntdot. mol-1 (1 cal = 4.18 J) for terminal A .cntdot. T compared to G .cntdot. T, C .cntdot. T, and T .cntdot. T mispairs, respectively, at 37.degree.C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that .DELTA..DELTA.G.degree. values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase .alpha.. The matched A .cntdot. T terminus is found to be extended .apprxeq. 200times faster than a G .cntdot. T mismatch and 1400 and 2500 times faster than C .cntdot. T and T .cntdot. T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37.degree.C, we find .DELTA..DELTA.G.degree. values of 2.6-3.7 kcal .cntdot. mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A .cntdot. T base pairs to be 5000 times greater than G .cntdot. T mispairs and 20,000 times greater than C .cntdot. T and T .cntdot. T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger .DELTA..DELTA.G.degree. values (4.3-4.9 kcal .cntdot. mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be > 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.