Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.
Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.
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DOI：
10.1093/nar/gks233
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发表时间：
2012-07
影响因子：
14.9
通讯作者：
Woodgate R
中科院分区：
文献类型：
--
作者：
Vaisman A;Kuban W;McDonald JP;Karata K;Yang W;Goodman MF;Woodgate R
大肠杆菌DNA聚合酶V的活性形式是一种多蛋白复合物（UmuD′2C-RecA-ATP），称为pol V Mut。在β/γ复合物和反式激活的RecA核蛋白丝RecA* 存在下，在SSB包被的单链环状DNA模板上观察到pol V Mut的体外最佳活性。值得注意的是，在这些条件下，野生型pol V Mut有效地将核糖核苷酸掺入DNA中。UmuC的“空间门”中的Y11 A取代进一步降低了pol V糖的选择性，并将pol V Mut转化为引物依赖性RNA聚合酶，该聚合酶能够以与DNA合成相当的持续合成能力合成长RNA。尽管有这些特性，Y11 A在体内仅促进低水平的自发诱变。虽然Y11 F取代对糖选择性的影响最小，但它导致自发诱变的增加。相比之下，F10 L取代增加了糖选择性和pol V Mut的总体保真度。分子模拟分析表明，L10的支链侧链撞击在Y11的苯环上，从而限制其运动，因此，牢固地关闭了空间门，这在野生型酶中无法以足够的严格性防止核糖核苷三磷酸的掺入。
The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD′2C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the ‘steric gate’ of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.
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影响因子：
3.8
作者：
Karata, Kiyonobu;Vaisman, Alexandra;Goodman, Myron F.;Woodgate, Roger
通讯作者：
Woodgate, Roger
影响因子：
14.9
作者：
Katafuchi, Atsushi;Sassa, Akira;Nohmi, Takehiko
通讯作者：
Nohmi, Takehiko
影响因子：
3.2
作者：
Kuban, Wojciech;Banach-Orlowska, Magdalena;Fijalkowska, Iwona J.
通讯作者：
Fijalkowska, Iwona J.
影响因子：
3.5
作者：
CHURCHWARD, G;BELIN, D;NAGAMINE, Y
通讯作者：
NAGAMINE, Y
影响因子：
3.2
作者：
DUTREIX, M;MOREAU, PL;DEVORET, R
通讯作者：
DEVORET, R