DYNAMIC-MODES OF THE FLIPPED-OUT CYTOSINE DURING HHAI METHYLTRANSFERASE-DNA INTERACTIONS IN SOLUTION

Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and Haem cytosine-5 methyltra nsferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes, H ere we studied the base dipping motions in the binary M.HhaI-DNA and t he ternary M.HhaI-DNA-cofactor systems in solution, Two 5-fluorocytosi nes were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides ups tream of the recognition sequence 5'-GCGC-3', The F-19 NMR spectra com bined with gel mobility data show that interaction with the enzyme ind uces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form l ocked in the enzyme active site, Addition of the cofactor analogue S-a denosyl-L-homocysteine greatly enhances the trapping of the target cyt osine in the catalytic site, Distinct dynamic modes of the target cyto sine have thus been identified along the reaction pathway, which inclu des novel base-dipping intermediates that were not. observed in previo us X-ray structures, The new data indicate that dipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the e nzyme in the opening of the DNA duplex.