Toggle Main Menu Toggle Search

Open Access padlockePrints

Determinants of Precatalytic Conformational Transitions in the DNA Cytosine Methyltransferase M.HhaI

Lookup NU author(s): Professor Bernard Connolly


Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


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.

Publication metadata

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


DOI: 10.1021/bi101446g


Altmetrics provided by Altmetric


Funder referenceFunder name
2008-25University of California