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Lookup NU author(s): Professor Bernard Connolly
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The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.
Author(s): Matje DM, Coughlin DF, Connolly BA, Dahlquist FW, Reich NO
Publication type: Article
Publication status: Published
Journal: Biochemistry
Year: 2011
Volume: 50
Issue: 9
Pages: 1465-1473
Print publication date: 13/01/2011
ISSN (print): 0006-2960
ISSN (electronic): 1520-4995
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/bi101446g
DOI: 10.1021/bi101446g
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