Self-Assembly of Escherichia coli MutL and Its Complexes with DNA
Format of Original
American Chemical Society
The Escherichia coli MutL protein regulates the activity of several enzymes, including MutS, MutH, and UvrD, during methyl-directed mismatch repair of DNA. We have investigated the self-association properties of MutL and its binding to DNA using analytical sedimentation velocity and equilibrium. Self-association of MutL is quite sensitive to solution conditions. At 25 °C in Tris at pH 8.3, MutL assembles into a heterogeneous mixture of large multimers. In the presence of potassium phosphate at pH 7.4, MutL forms primarily stable dimers, with the higher-order assembly states suppressed. The weight-average sedimentation coefficient of the MutL dimer in this buffer (s̅20,w) is equal to 5.20 ± 0.08 S, suggesting a highly asymmetric dimer (f/fo = 1.58 ± 0.02). Upon binding the nonhydrolyzable ATP analogue, AMPPNP/Mg2+, the MutL dimer becomes more compact (s̅20,w = 5.71 ± 0.08 S; f/fo = 1.45 ± 0.02), probably reflecting reorganization of the N-terminal ATPase domains. A MutL dimer binds to an 18 bp duplex with a 3′-(dT20) single-stranded flanking region, with apparent affinity in the micromolar range. AMPPNP binding to MutL increases its affinity for DNA by a factor of ∼10. These results indicate that the presence of phosphate minimizes further MutL oligomerization beyond a dimer and that differences in solution conditions likely explain apparent discrepancies in previous studies of MutL assembly.