Self-association Equilibria of Escherichia coli UvrD Helicase Studied by Analytical Ultracentrifugation

Abstract

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 20 mM to 200 mM decreases the dimerization equilibrium constant ( L 20) 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 ( L 40) is 5.11(±0.80) μM −3 at 20 mM NaCl, but decreases so that it is not measurable at 200 mM NaCl. At 500 mM NaCl, only UvrD monomers are detectable. Increasing [glycerol] over the range from 20% to 40% (v/v) decreases both L 20 and L 40. 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 L 20 or L 40 significantly, and does not induce the formation of additional assembly states. We also investigated the stoichiometry of UvrD binding to a 3′-(dT) 20-18 bp 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.