Fidelity and error specificity of the alpha catalytic subunit of Escherichia coli DNA polymerase III
Fidelity and error specificity of the alpha catalytic subunit of Escherichia coli DNA polymerase III
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DOI：
10.1074/jbc.271.31.18947
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发表时间：
1996-08-02
影响因子：
4.8
通讯作者：
Schaaper, RM
中科院分区：
文献类型：
--
作者：
Mo, JY;Schaaper, RM
大肠杆菌 DNA 聚合酶 III 全酶是主要负责大肠杆菌染色体复制的复制酶。这个过程的发生精度很高，每轮复制每个碱基对发生的错误少于 10(-9) 到 10(-10) 个。作为了解该过程高保真度机制的第一步，我们纯化了聚合酶 III α 催化亚基，使其不含核酸外切酶活性，并分析了其体外保真度。我们采用了新开发的缺口填充测定法，使用基因座基因的 N 端 250 个碱基作为正向突变靶标。当跨该靶点进行合成时，α 亚基产生突变的频率为 0.6%。 DNA测序显示，体外产生的突变体主要由移码突变组成，尽管也观察到一些碱基替换。发生的移码超过背景120倍以上，主要由-1缺失组成。其中，约80%是带有嘧啶5'-邻近的嘌呤模板碱基的缺失。这些结果表明，α亚基(i)具有相对较低的从错误掺入的碱基延伸的能力，解释了观察到的碱基取代的低水平，以及(ii)在错误掺入的碱基与下一个（互补的）模板碱基错配后具有相对较高的延伸能力，解释了高水平的移码突变。该模型得到了一项实验的支持，其中需要 α 亚基从序列环境中的末端错配启动 DNA 合成，从而允许在下一个模板碱基上滑动。在该反应的产物中，移码数量超过碱基对取代数量的 70 倍以上。与体内突变谱的比较表明，pol III 辅助因子可能在调节 DNA 合成保真度方面发挥重要作用。
Escherichia coli DNA polymerase III holoenzyme is the replicative enzyme primarily responsible for the duplication of the E. coli chromosome. This process occurs with high accuracy, less than 10(-9) to 10(-10) errors being committed per base pair per round of replication. As a first step in understanding the mechanisms responsible for the high fidelity of this process, we have purified the polymerase III alpha catalytic subunit, free of exonuclease activity, and analyzed its fidelity in vitro. We employed a newly developed gap-filling assay using the N-terminal 250 bases of the loci gene as a forward mutational target. When synthesizing across this target, alpha subunit produced mutations at a frequency of 0.6%. DNA sequencing revealed that the mutants created in vitro consisted mostly of frameshift mutations, although some base substitutions were also observed, The frameshifts, occurring at more than 120-fold above the background, consisted largely of -1 deletions. Among them, about 80% were the deletion of a purine template base with a pyrimidine 5'-neighbor, These results suggest that the alpha subunit (i) has a relatively low ability to extend from misincorporated bases, accounting for the low level of observed base substitutions, and (ii) has a relatively high capability of extension after misalignment of a misincorporated base on the next (complementary) template base, accounting for the high level of frameshift mutations. This model is supported by an experiment in which alpha subunit was required to initiate DNA synthesis from a terminal mispair in a sequence context that allowed slippage on the next template base. Among the products of this reaction, frameshifts outnumbered base pair substitutions by greater than 70-fold. A comparison to in vivo mutational spectra suggests that the pol III accessory factors may play a major role in modulating the fidelity of DNA synthesis.