title: Self-assembly of Escherichia coli MutL and its complexes with DNA.
abstract: 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 ( ̅s(20,w)) is equal to 5.20 ± 0.08 S, suggesting a highly asymmetric dimer (f/f(o) = 1.58 ± 0.02). Upon binding the nonhydrolyzable ATP analogue, AMPPNP/Mg(2+), the MutL dimer becomes more compact ( ̅s(20,w) = 5.71 ± 0.08 S; f/f(o) = 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'-(dT(20)) 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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, Missouri 63110-1093, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Niedziela-Majka
authors.forename: Nasib K
authors.initials: NK
authors.lastname: Maluf
authors.forename: Edwin
authors.initials: E
authors.lastname: Antony
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D000070956:MutL Proteins; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D010710:Phosphates; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins