Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation

The Escherichia coli UvrD protein (helicase II) is an SF1 superfamily helicase required for methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized quantitatively the self-assembly equilibria of the UvrD protein as a function of [NaCl], [glycerol], and temperature (5-35°C; pH 8.3) using analytical sedimentation velocity and equilibrium techniques, and find that UvrD self-associates into dimeric and tetrameric species over a range of solution conditions (t≤25°C). Increasing [NaCl] from 20mM to 200mM decreases the dimerization equilibrium constant (L₂₀) from 2.33(±0.30)μM^-1 to 0.297(±0.006)μM^-1 (pH 8.3, 20% (v/v) glycerol, 25°C). The overall tetramerization equilibrium constant (L₄₀) is 5.11(±0.80)μM^-3 at 20mM NaCl, but decreases so that it is not measurable at 200mM NaCl. At 500mM NaCl, only UvrD monomers are detectable. Increasing [glycerol] over the range from 20% to 40% (v/v) decreases both L₂₀ and L₄₀. We find no evidence for hexamer formation, although a species consistent in size with an octamer is detected at 35°C. Inclusion of either ADP or ATPγS does not affect either L₂₀ or L₄₀ significantly, and does not induce the formation of additional assembly states. We also investigated the stoichiometry of UvrD binding to a 3′-(dT)₂₀-18bp DNA substrate by sedimentation equilibrium. At saturating concentrations of UvrD, three UvrD monomers can bind to the DNA substrate, although only two UvrD monomers are required to form a processive helicase complex. When the total DNA substrate concentration is about twofold greater than the total UvrD concentration, the vast majority of the DNA is bound by a single UvrD monomer.