DNA polymerase β:: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes
DNA polymerase β:: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes
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DOI：
10.1042/bj3310079
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发表时间：
1998-04-01
影响因子：
4.1
通讯作者：
Tsai, MD
中科院分区：
文献类型：
--
作者：
Ahn, JW;Kraynov, VS;Tsai, MD
使用预稳态动力学分析来比较DNA聚合酶β（Pol β）用于单碱基缺口填充和常规双链体DNA合成的催化性质。对于所有四种可能的正确碱基对和十二种可能的不正确碱基对，用单核苷酸缺口DNA底物测定聚合速率（k（pol））和dNTP的表观平衡解离常数（K-d），并将结果与先前用无缺口引物/模板双链体DNA底物获得的结果进行比较。单核苷酸缺口DNA的使用导致dNTP的Kd显著降低。虽然k（pol）的影响不大，但由于K-d的降低，催化效率k（pol）/K-d显著增加。相反，对于不正确的dNTP掺入，使用单核苷酸缺口DNA底物并不明显影响dNTP的Kd，但导致k（pol）因此，Pol β的保真度不受使用单核苷酸缺口DNA底物的显著影响，此外，我们表明，在进行性聚合条件下，由于DNA解离速率降低，Pol β的持续合成能力在间隙填充合成中增加。最后，用单核苷酸缺口DNA底物也观察到化学前的限速构象变化步骤，然而，用双链体DNA底物观察到的前面的快速构象变化没有被清楚地检测到，一个可能的原因是在具有缺口DNA的复合物中，Pol β的8 kDa N-末端结构域已经以闭合构象存在。胰蛋白酶消化实验支持这一解释。
Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (k(pol)) and the apparent equilibrium dissociation constant of dNTP (K-d) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates, For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the K-d of dNTP. Although k(pol) was little affected, the catalytic efficiency k(pol)/K-d increased significantly owing to the decreases in K-d. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the K-d of dNTP appreciably but caused the k(pol) (and thus k(pol)/K-d) for incorrect dNTP incorporation to increase, As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates, In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filing synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed, However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected, A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.