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 ...
Most damage induced mutagenesis in Escherichia coli is dependent upon the [Formula: see text] 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 4L of bacterial culture. Rather than over producing the UmuC protein, it was expressed at low basal levels, while [Formula: see text] 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 ( [Formula: see text] ), which was efficiently recruited to a primer terminus by SSB.



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