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

Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'C-2 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 UmuD'(2) was expressed in trans from a high copy-number plasmid with an inducible promoter. We have also developed strategies to purify the beta-clamp and gamma-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 beta/gamma-complex and a RecA nucleoprotein filament (RecA*) formed in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (UmuD'C-2 . RecA . ATP), which was efficiently recruited to a primer terminus by SSB. Published by Elsevier B.V.