DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.
DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.
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DOI：
10.1021/bi9527202
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发表时间：
1996-06
期刊：
影响因子：
2.9
通讯作者：
B. G. Werneburg;J. Ahn;X. Zhong;R. Hondal;V. Kraynov;M. Tsai
中科院分区：
文献类型：
--
作者：
B. G. Werneburg;J. Ahn;X. Zhong;R. Hondal;V. Kraynov;M. Tsai
DNA聚合酶β（Polbeta）是最小和最不复杂的DNA聚合酶。这种酶的结构已经很清楚，但对其催化性能，特别是持续合成能力和保真度知之甚少。通过pol β将单个核苷酸掺入短的25/45寡核苷酸引物-模板的预稳态分析用于定义聚合酶的动力学参数。此外，核苷酸类似物和位点特异性突变体，沿着结构分析，用于探测pol β的结构-功能关系。已经获得了几个重要的发现：（i）聚合酶β的催化是进行性的，并且在预稳态条件下显示初始爆发，但是与其他聚合酶相比，其进行性较差。(ii)相对于其他聚合酶，pol β的保真度也较低。(iii)在预稳态条件下，基于低硫代效应（4.3），化学步骤似乎仅部分限速，定义为kpol（dNTP）/kpol（dNTP α S）。硫代效应增加到9为不正确的核苷酸的掺入。这些结果与底物诱导的构象变化的存在是一致的，这也是部分限速的。(iv)野生型和突变酶的二维NMR谱之间的比较表明，在位置283的突变没有显着扰动的酶的结构。突变体的构象稳定性也未受干扰。因此，R283对酶的整体结构并不重要。(v)R283 A和R283 K突变体的动力学分析结果表明，聚合物β的R283与模板之间的氢键对催化作用是重要的。R283 A和R283 K突变体均显示催化效率降低约1倍。200相对于野生型pol β。就T-G错配对与T-A正确配对而言，突变体的忠诚度也低2-4倍。然而，扰动可能发生在暗示的构象步骤和化学步骤，因为突变体对正确和不正确核苷酸的硫代效应与WT pol β的硫代效应相似。
DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.