Self-Assembly of Escherichia coli MutL and Its Complexes with DNA

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• 作者：Anita Niedziela-Majka ; Nasib K. Maluf ; Edwin Antony ; Timothy M. Lohman
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：September 20, 2011
• 年：2011
• 卷：50
• 期：37
• 页码：7868-7880
• 全文大小：621K
• 年卷期：v.50,no.37(September 20, 2011)
• ISSN：1520-4995

文摘

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 掳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 (s̅_20,w) is equal to 5.20 卤 0.08 S, suggesting a highly asymmetric dimer (f/f_o = 1.58 卤 0.02). Upon binding the nonhydrolyzable ATP analogue, AMPPNP/Mg²⁺, the MutL dimer becomes more compact (s̅_20,w = 5.71 卤 0.08 S; f/f_o = 1.45 卤 0.02), probably reflecting reorganization of the N-terminal ATPase domains. A MutL dimer binds to an 18 bp duplex with a 3鈥?(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.