title: 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 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.
authors.affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
authors.forename: Kiyonobu
authors.initials: K
authors.lastname: Karata
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vaisman
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D005591:Chemical Fractionation / Q000379:methods*; D003067:Coenzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D009154:Mutation; D011938:Rec A Recombinases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D003067:Coenzymes; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases