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

Abstract:

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.

Polymerases:

Topics:

Source / Purification

Status:

new topics/pols set partial results complete validated

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