Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex.
We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.
Hopkins
Ben B
BB
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
Reich
Norbert O
NO
D020816:Amino Acid Motifs; D016880:Anisotropy; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry; D004720:Endonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008956:Models, Chemical; D015233:Models, Statistical; D009690:Nucleic Acid Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004720:Endonucleases; D045647:DNA Glycosylases