Dysfunctional proofreading in the Escherichia coli DNA polymerase III core
Dysfunctional proofreading in the Escherichia coli DNA polymerase III core
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DOI：
10.1042/bj20040660
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发表时间：
2004-12-01
影响因子：
4.1
通讯作者：
Perrino, FW
中科院分区：
文献类型：
--
作者：
Lehtinen, DA;Perrino, FW
epsilon-亚基包含3‘ -> - 5’校对外切酶的催化位点，该酶在DNA pol III （DNA聚合酶III）核心中起作用，编辑被a亚基DNA pol错插入的核苷酸。一种新的诱变策略被用来鉴定23个在体内表现出突变表型的dnaQ等位基因。14个epsilon突变体被纯化，这些蛋白表现出3‘ - bbb50 ’外切酶活性，其活性范围为野生型epsilon蛋白的32%至155%，而纯化的MutD5蛋白表现出2%的活性。DNA pol III核心酶由14个epsilon突变体中的11个组成，在体外DNA合成过程中显示出更高的误差率。纯化的epsilon突变体与α -亚基和α -亚基的相互作用通过凝胶过滤层析和核酸外切酶刺激实验进行检测，并通过测定聚合酶/核酸外切酶比来鉴定具有催化活性的epsilon511 （I170T/V215A）突变体，该突变体在DNA pol III核心中具有功能失调的校对功能。epsilon511突变体与a亚基密切相关，但在α -epsilon511复合体中，epsilon511的外切酶活性没有受到刺激。添加theta-亚基生成α - epsilon511 -theta DNA pol III核心部分恢复了对epsilon511外切酶的刺激，表明theta-亚基在协调α -epsilon聚合酶-外切酶相互作用中的作用。与野生型DNA pol III核心相比，α -epsilon511-theta DNA pol III核心的聚合酶/外切酶比高出3.5倍，进一步表明α -epsilon511-theta复合物的校对功能不正常。因此，epsilon511突变体具有野生型3‘ - bbb50 ’外切酶活性，并与α -亚基和β -亚基物理结合，产生具有校对缺陷的DNA pol III酶。
The epsilon-subunit contains the catalytic site for the 3' --> 5' proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the a-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the epsilon mutants were purified, and these proteins exhibited 3' --> 5' exonuclease activities that ranged from 32 % to 155 % of the activity exhibited by the wild-type epsilon protein, in contrast with the 2 % activity exhibited by purified MutD5 protein. DNA pol III core euzymes constituted with 11 of the 14 epsilon mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified epsilon mutants with the alpha- and theta-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active epsilon511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The epsilon511 mutant associated tightly with the a-subunit, but the exonuclease activity of epsilon511 was not stimulated in the alpha-epsilon511 complex. Addition of the theta-subunit to generate the alpha-epsilon5 11-theta DNA pol III core partially restored stimulation of the epsilon511 exonuclease, indicating a role for the theta-subunit in coordinating the alpha-epsilon polymerase-exonuclease interaction. The alpha-epsilon511-theta DNA pol III core exhibited a 3.5-fold higher polymerase/ exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the alpha-epsilon511-theta complex. Thus the epsilon511 mutant has wild-type 3' --> 5' exonuclease activity and associates physically with the alpha- and theta-subunits to generate a proofreading-defective DNA pol III enzyme.