KINETIC MECHANISM OF DNA-POLYMERASE-I (KLENOW)
KINETIC MECHANISM OF DNA-POLYMERASE-I (KLENOW)
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DOI：
10.1021/bi00399a057
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发表时间：
1987-12-15
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：
KUCHTA, RD;MIZRAHI, V;BENKOVIC, SJ
用确定序列的短DNA寡聚体确定了大肠杆菌DNA聚合酶I（KF）Klenow片段催化DNA聚合的最小动力学方案。该方案的关键特征是使三元KF·DNan·dNTP和KF·DNan+1·PPi复合物相互转化的最小两步序列。速率不受实际聚合的限制，而是受单独步骤的限制，这在确保保真度方面可能是重要的[Mizrahi，V.，亨利河N.，Marlier，J.F.，约翰逊，K. A.和Benkovic，S. J.（1985）Biochemistry 24，4010-4018]。这一序列的证据是由双相动力学的观察提供的单周转焦磷酸解实验（聚合的微观逆转）。然后，数据分析提供了内部平衡常数的估计。DNA，dNTP，和PPi从各种二元和三元复合物的解离进行了测量分区（同位素捕获）实验。DNA从KF解离的速率常数是序列依赖性的，并且在非进行性DNA合成期间是速率限制性的。单营业额（两个方向）和同位素捕获实验的组合提供了足够的信息，允许定量评价特定DNA序列的动力学方案。
The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.cntdot.DNAn.cntdot.dNTP and KF.cntdot.DNAn+1.cntdot.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A. and Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments(the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.