Simple and efficient purification of Escherichia coli DNA polymerase V: Cofactor requirements for optimal activity and processivity in vitro

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• 作者：Kiyonobu Karata^a ; ¹ ; Alexandra Vaisman^a ; Myron F. Goodman^b ; ^c ; Roger Woodgate^a ; ^{woodgate@nih.gov}
• 关键词：UmuC ; UmuD&prime ; SOS mutagenesis ; Y-family DNA polymerase ; Mutagenesis ; Translesion DNA synthesis
• 刊名：DNA Repair
• 出版年：2012
• 期刊代码：121_15687864
• 类别：ch
• 出版时间：1 April, 2012
• 卷：11
• 期：4
• 页码：431-440
• 文件大小：1371 K

摘要

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 尾-clamp and 纬-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 尾/纬-complex and a RecA nucleoprotein filament (RecA*) formed in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (), which was efficiently recruited to a primer terminus by SSB.