The Base Substitution and Frameshift Fidelity ofEscherichia coli DNA Polymerase III Holoenzyme in Vitro *
The Base Substitution and Frameshift Fidelity ofEscherichia coli DNA Polymerase III Holoenzyme in Vitro *
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DOI：
10.1074/jbc.273.36.23575
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发表时间：
1998-09
期刊：
影响因子：
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通讯作者：
P. Pham;M. Olson;C. McHenry;R. Schaaper
中科院分区：
文献类型：
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作者：
P. Pham;M. Olson;C. McHenry;R. Schaaper
我们研究了野生型和校对受损mutD5菌株的大肠杆菌DNA聚合酶III全酶的体外保真度。外切酶实验表明，mutD5全酶的3 ‘→5 ’外切酶活性降低了30 - 50倍。在跨lacI前向突变靶点的间隙填充合成过程中测定了保真度。两种酶在低dNTP浓度（10 ~ 50 μM）下的误差率最低，在高dNTP浓度（1000 μM）下错误率最高。mutD5校对缺陷仅使错误率增加了3 - 5倍。除了碱基取代外，这两种酶都产生了高水平的（−1）移码突变。碱基置换主要是C→T、G→T和G→C，但dNTP池失衡表明，这些可能反映了与受损模板碱基相反的错结合，而T→C、G→A和C→T的转变代表了正常的聚合酶iii介导的碱基·碱基错配。频繁的（- 1）移码突变不是由直接滑移引起的，而是通过一种涉及“误合并加滑移”的机制产生的。对野生型和mutD5全酶在M13体内复制过程中保真度的测量显示，在移码错误频率和校对程度方面，体内保真度和体外保真度存在显著差异。
We have investigated the in vitrofidelity of Escherichia coli DNA polymerase III holoenzyme from a wild-type and a proofreading-impaired mutD5 strain. Exonuclease assays showed the mutD5 holoenzyme to have a 30–50-fold reduced 3′→5′-exonuclease activity. Fidelity was assayed during gap-filling synthesis across the lacI dforward mutational target. The error rate for both enzymes was lowest at low dNTP concentrations (10–50 μM) and highest at high dNTP concentration (1000 μM). The mutD5 proofreading defect increased the error rate by only 3–5-fold. Both enzymes produced a high level of (−1)-frameshift mutations in addition to base substitutions. The base substitutions were mainly C→T, G→T, and G→C, but dNTP pool imbalances suggested that these may reflect misincorporations opposite damaged template bases and that, instead, T→C, G→A, and C→T transitions represent the normal polymerase III-mediated base·base mispairs. The frequent (−1)-frameshift mutations do not result from direct slippage but may be generated via a mechanism involving “misincorporation plus slippage.” Measurements of the fidelity of wild-type and mutD5 holoenzyme during M13 in vivo replication revealed significant differences between the in vivo and in vitro fidelity with regard to both the frequency of frameshift errors and the extent of proofreading.