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

John R. Horton, Gary Ratner, Nilesh K. Banavali, Niu Huang, Yongseok Choi, Martin A. Maier, Victor E. Marquez, Alexander D. MacKerell, Xiaodong Cheng

Research output: Contribution to journalArticlepeer-review

45 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)3877-3886
Number of pages10
JournalNucleic acids research
Issue number13
Publication statusPublished - 2004
Externally publishedYes

Bibliographical note

Funding Information:
X.C. is grateful to Dr Richard J. Roberts (New England Biolabs) and Dr Robert M. Blumenthal (Medical College of Ohio) for their continued support and encouragement. We thank Susan Sunay (Emory University) for help with purification and crystallization, Annie Heroux (Brookhaven National Laboratory) for help with X-ray data collection at beamline X26C in the facilities of the National Synchrotron Light Source. Work was supported in part by National Institutes of Health Grants GM49245 to X.C. and GM51501 to A.D.M.

ASJC Scopus subject areas

  • Genetics


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