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

DNA Repair (Amst). 2012 Apr 1;11(4):431-40. doi: 10.1016/j.dnarep.2012.01.012. Epub 2012 Feb 15.


Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'(2)C 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)C 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 (UmuD'(2)C · RecA · ATP), which was efficiently recruited to a primer terminus by SSB.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Chemical Fractionation / methods*
  • Coenzymes / metabolism*
  • DNA, Single-Stranded / biosynthesis
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism*
  • Enzyme Assays
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification*
  • Escherichia coli Proteins / metabolism*
  • Exodeoxyribonucleases / metabolism
  • Mutation
  • Rec A Recombinases / metabolism
  • Sequence Homology, Amino Acid
  • Solubility


  • Coenzymes
  • DNA, Single-Stranded
  • Escherichia coli Proteins
  • Adenosine Triphosphate
  • UmuC protein, E coli
  • Rec A Recombinases
  • DNA polymerase V, E coli
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases