MINIMAL KINETIC MECHANISM FOR MISINCORPORATION BY DNA-POLYMERASE-I (KLENOW FRAGMENT)
MINIMAL KINETIC MECHANISM FOR MISINCORPORATION BY DNA-POLYMERASE-I (KLENOW FRAGMENT)
复制标题
DOI：
10.1021/bi00153a016
复制
发表时间：
1992-09-29
期刊：
影响因子：
2.9
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
--
作者：
EGER, BT;BENKOVIC, SJ
DNA聚合酶I的Klenow片段[KF(exo+)]将单核苷酸(DATP)错误结合到短DNA引物/模板(9/20-mer)中的最小动力学机制已在先前发表[Kuchta，R.D.，Benkovic，P.，&Benkovic，S.J.(1988)BioChemical 27,6716-6725]。本文对这一机制进行了改进。在存在过量KF(exo+)的单个错误核苷酸(DATP)的情况下，正确核苷酸掺入(DTTP)的稳态前测量表明，dATP在化学之前的两个步骤中与KF(exo+)-9/20-聚体结合。用(Alphas)dATP取代dATP产生了相同的两步结合动力学，消除了核苷酸结合作为对误掺入率的元素影响的原因。三元物种[KF‘(exo+)-9A/20-mer-PP(I)]的焦磷酸盐释放发生在限速构象变化之后，该物种通过内部焦磷分解等分成核苷酸或错配产物。9A/20-聚体从中心三元络合物(KF‘-9A/20-MET-PP(I))20-Mer-PP(I)的解离速率与核酸外切作用相比可以忽略不计。对错误结合的DNA产物(9A/20-mer)进行焦磷酸分解，结合测量dATP错误结合的速率，可以确定dATP错误结合的总体平衡常数，并提供与正确结合所测得的值相似的值。逐步比较Klenow片段催化的聚合反应的正确和不正确的核苷酸掺入，强调酶的复制保真度的主要来源来自实际化学步骤的区分，以及由于三元错配产物复合体上核酸外切酶活性的增加，因为它通过转换序列的速度较慢。
The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27,6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alphaS)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PP(i)] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-met-PP(i)) 20-mer-PP(i)) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.