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J. Biol. Chem., Vol. 279, Issue 30, 31419-31428, July 23, 2004

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The Coupling of Tight DNA Binding and Base Flipping

IDENTIFICATION OF A CONSERVED STRUCTURAL MOTIF IN BASE FLIPPING ENZYMES^*

R. August Estabrook, Rebecca Lipson, Ben Hopkins, and Norbert Reich

From the Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106

Val¹²¹ is positioned immediately above the extrahelical cytosine in HhaI DNA C⁵-cytosine methyltransferase, and replacement with alanine dramatically interferes with base flipping and catalysis. DNA binding and k_cat are decreased 10⁵-fold for the Val¹²¹ Ala mutant that has a normal circular dichroism spectrum and AdoMet affinity. The magnitude of this loss of function is comparable with removal of the essential catalytic Cys⁸¹. Surprisingly, DNA binding is completely recovered (increase of 10⁵-fold) with a DNA substrate lacking the target cytosine base (abasic). Thus, interfering with the base flipping transition results in a dramatic loss of binding energy. Our data support an induced fit mechanism in which tight DNA binding is coupled to both base flipping and protein loop rearrangement. The importance of the proximal protein segment (His¹²⁷–Thr¹³²) in maintaining this critical interaction between Val¹²¹ and the flipped cytosine was probed with single site alanine substitutions. None of these mutants are significantly altered in secondary structure, AdoMet or DNA affinity, k_methylation, k_inactivation, or k_cat. Although Val¹²¹ plays a critical role in both extrahelical base stabilization and catalysis, its position and mobility are not influenced by individual residues in the adjacent peptide region. Structural comparisons with other DNA methyltransferases and DNA repair enzymes that stabilize extrahelical nucleotides reveal a motif that includes a positively charged or polar side chain and a hydrophobic residue positioned adjacent to the target DNA base and either the 5'- or 3'-phosphate.

Received for publication, March 16, 2004 , and in revised form, May 5, 2004.

^* This work was supported by National Institutes of Health Grant GM 463333 and National Science Foundation Grant MCB-9983125 (to N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel:. 805-893-8368; Fax: 805-893-4120; E-mail: reich{at}chem.ucsb.edu.

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