Fidelity of Escherichia coli DNA polymerase III holoenzyme - The effects of beta,gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies
Fidelity of Escherichia coli DNA polymerase III holoenzyme - The effects of beta,gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies
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DOI：
10.1074/jbc.272.44.27919
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Goodman, MF
中科院分区：
文献类型：
--
作者：
Bloom, LB;Chen, XL;Goodman, MF
测量大肠杆菌 DNA 聚合酶 III (pol III) 的保真度，并使用凝胶动力学测定评估 β、gamma 持续合成能力和 epsilon 校对亚基的影响，Pol III 全酶合成保真度极高的 DNA，将 dTMP、dAMP 和 dGMP 与模板 G 靶标相对，效率 f(inc) = 5.6 x 10(-6)，分别为 4.2 x 10(-7) 和 7 x 10(-7)。当 C 和 A 分别位于模板目标 G 下游一个碱基时，由于“dNTP 稳定”DNA 错位机制，dGMP.G 和 dTMP.G 错误掺入效率提高了 3.2 x 10(-5) 和 5.8 x 10(-4)。至少 92% 的错误插入核苷酸被 pol III 全酶切除缺少下一个正确的“救援”核苷酸，随着救援 dNTP 浓度的增加，pol III 全酶的保真度最多降低 8 倍，因为错配引物末端的校对在与下一个正确核苷酸的掺入竞争中减少，与 pol III 全酶相比，无法校对的 α 全酶的保真度高出 47 倍、32 倍和 13 倍dGMP.G、dTMP.G 和 dAMP.G 错配的错误掺入率。 β、γ 复合物和下游核苷酸对催化 α 亚基的保真度几乎没有影响，“附录”中介绍了当发生多次聚合酶-DNA 相遇时凝胶动力学保真度测定的分析（参见 Fygenson, D. K. 和 Goodman, M. F. (1997) J. Biol. Chem, 272, 27931-27935（随附纸））。
The fidelity of Escherichia coli DNA polymerase III (pol III), is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay, Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f(inc) = 5.6 x 10(-6), 4.2 x 10(-7) and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a ''dNTP-stabilized'' DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of mis-inserted nucleotides are excised by pol III holoenzyme in the absence of a next correct ''rescue'' nucleotide, As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide, Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit, An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the ''Appendix'' (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem, 272, 27931-27935 (accompanying paper)).