Document Type

Article

Language

eng

Format of Original

13 p.

Publication Date

9-2011

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

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 (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.

Comments

Accepted version. Biochemistry, Vol. 50, No. 37 (September 2011): 7868-7880. DOI. © 2011 American Chemical Society Publications. Used with permission.

Edwin Antony was affiliated with Washington University at time of publication.

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