Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro.
Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro.
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DOI：
10.1016/j.dnarep.2012.01.012
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发表时间：
2012-04-01
期刊：
影响因子：
3.8
通讯作者：
Woodgate, Roger
中科院分区：
文献类型：
--
作者：
Karata, Kiyonobu;Vaisman, Alexandra;Goodman, Myron F.;Woodgate, Roger
大肠杆菌中的大多数损伤诱导突变依赖于 蛋白质复合物，其包含DNA聚合酶V（pol V）。pol V的生物化学表征受到这样的事实的阻碍，即该酶是众所周知的难以纯化，主要是因为过量产生的UmuC是不溶性的。在这里，我们报告了一个简单而有效的协议，快速纯化毫克量的pol V从只有4升的细菌培养。UmuC蛋白不是过量产生，而是以低基础水平表达， 从具有诱导型启动子的高拷贝数质粒反式表达。我们还开发了纯化β-夹和γ-夹加载剂而不含污染聚合酶的策略。使用这些高度纯化的蛋白质，我们确定了pol V体外最佳活性的辅因子要求，并发现pol V在β/γ-复合物和反式形成的RecA核蛋白丝（RecA*）存在下对SSB包被的环状DNA模板显示出稳健的活性。（· RecA · ATP），其被SSB有效募集到引物末端。
Most damage induced mutagenesis in Escherichia coli is dependent upon the protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. Here, we report a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just 4 L of bacterial culture. Rather than over producing the UmuC protein, it was expressed at low basal levels, while was expressed in trans from a high copy-number plasmid with an inducible promoter. We have also developed strategies to purify the β-clamp and γ-clamp loader free from contaminating polymerases. Using these highly purified proteins, we determined the cofactor requirements for optimal activity of pol V in vitro and found that pol V shows robust activity on an SSB-coated circular DNA template in the presence of the β/γ-complex and a RecA nucleoprotein filament (RecA*) formed in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut ( · RecA · ATP), which was efficiently recruited to a primer terminus by SSB.
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影响因子：
5.6
作者：
McDonald, JP;Peat, TS;Woodgate, R
通讯作者：
Woodgate, R
影响因子：
5.6
作者：
Fujii, S;Gasser, W;Fuchs, RP
通讯作者：
Fuchs, RP
DOI：
10.1073/pnas.93.19.10291
发表时间：
1996-09-17
影响因子：
11.1
作者：
Frank, EG;Ennis, DG;Woodgate, R
通讯作者：
Woodgate, R
影响因子：
3.5
作者：
CHURCHWARD, G;BELIN, D;NAGAMINE, Y
通讯作者：
NAGAMINE, Y
DOI：
10.1073/pnas.81.15.4622
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BLANAR, MA;SANDLER, SJ;CLARK, AJ
通讯作者：
CLARK, AJ