Book of Abstracts: Albany 2005
Nucleotide Flipping: Mechanisms and Specificity Determinants
We identified a motif in the bacterial DNA cytosine methyltransferase M.HhaI, which is important for nucleotide flipping and extrahelical stabilization. This motif includes a positively charged (Arg165 for M.HhaI) or polar side chain, a small hydrophobic residue positioned in van der Waals contact with the target base (Val121 for M.HhaI), which is in close proximity to the phosphate on either the 5' or 3' side of the target base. Structural and functional studies of this motif in M.HhaI show that this motif is absolutely essential for the correct assembly of the extrahelical base. The structural studies of a mutant:DNA complex show the target cytosine positioned 30-40° into the major groove of the DNA, supporting a major groove trajectory for the target base. The motif is conserved in DNA cytosine and adenine methyltransferases and DNA repair enzymes. M.HhaI mutants designed to test the importance of the highly flexible loop in nucleotide flipping show significant increases in substrate specificity, mediated through the destabilization of reaction intermediates. Crystallographic analysis of these mutants shows this stabilization is caused by a cavity created at the site of substitution. These results suggest that nucleotide flipping is a specificity determinant.
We also identified a mutant of the DNA adenine methyltransferase, M.EcoRI, which unlike the wild type enzyme, does not bend its cognate site and which causes the nucleotide flipping rate to be slowed down 2000 fold. This mutant is also increased in specificity. M.EcoRI, in addition to bending its target by 50° and inducing nucleotide flipping, intercalates several amino acids into the DNA. The binding, bending, intercalation and flipping transitions are nearly concerted for the cognate site; similar measurements with non-cognate sites shows that these conformational transitions contribute to the enzyme?s specificity.
Norbert O. Reich*
Department of Chemistry and Biochemistry