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Nucleic Acids Research 2004 32(13):3877-3886; doi:10.1093/nar/gkh701
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Published online 23 July 2004

Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved

Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase

John R. Horton1, Gary Ratner1,2, Nilesh K. Banavali3, Niu Huang3, Yongseok Choi4, Martin A. Maier5, Victor E. Marquez4, Alexander D. MacKerell, Jr3 and Xiaodong Cheng1,*

1 Department of Biochemistry and 2 Graduate Program in Biochemistry, Cell, and Development Biology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA, 3 Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA, 4 Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, 376 Boyles Street, Bldg 376, Rm 104, Frederick, MD 21702-1201, USA and 5 Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, CA 92008, USA

* To whom correspondence should be addressed. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: xcheng{at}emory.edu
Present addresses: Nilesh K. Banavali, Department of Biochemistry and Structural Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
Niu Huang, Department of Biopharmaceutical Sciences, UCSF, 600, 16th Street, Suite N474E, San Francisco, CA 94143, USA

Received May 4, 2004; Revised June 16, 2004; Accepted June 24, 2004

Rotation of a DNA or RNA nucleotide out of the double helix and into a protein pocket (‘base flipping’) is a mechanistic feature common to some DNA/RNA-binding proteins. Here, we report the structure of HhaI methyltransferase in complex with DNA containing a south-constrained abasic carbocyclic sugar at the target site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the locked south pseudosugar appears to be trapped in the middle of the flipping pathway via the DNA major groove, held in place primarily through Van der Waals contacts with a set of invariant amino acids. Molecular dynamics simulations indicate that the structural stabilization observed with the south-constrained pseudosugar will not occur with a north-constrained pseudosugar, which explains its lowered binding affinity. Moreover, comparison of structural transitions of the sugar and phosphodiester backbone observed during computational studies of base flipping in the M.HhaI–DNA–AdoHcy ternary complex indicate that the south-constrained pseudosugar induces a conformation on the phosphodiester backbone that corresponds to that of a discrete intermediate of the base-flipping pathway. As previous crystal structures of M.HhaI ternary complex with DNA displayed the flipped sugar moiety in the antipodal north conformation, we suggest that conversion of the sugar pucker from south to north beyond the middle of the pathway is an essential part of the mechanism through which flipping must proceed to reach its final destination. We also discuss the possibility of the south-constrained pseudosugar mimicking a transition state in the phosphodiester and sugar moieties that occurs during DNA base flipping in the presence of M.HhaI.


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