Stopped-flow and mutational analysis of base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase.
By stopped-flow kinetics using 2-aminopurine as a probe to detect base flipping, we show here that base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase (MTase) is a biphasic process: target base flipping is very fast (k(flip)>240 s(-1)), but binding of the flipped base into the active site pocket of the enzyme is slow (k=0.1-2 s(-1)). Whereas base flipping occurs in the absence of S-adenosyl-l-methionine (AdoMet), binding of the target base in the active site pocket requires AdoMet. Our data suggest that the tyrosine residue in the DPPY motif conserved in the active site of DNA-(adenine-N6)-MTases stacks to the flipped target base. Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base. The exchange of Ser188 located in a loop next to the active center by alanine led to a seven- to eightfold reduction of k(flip), which was also reduced with substrates having altered GATC recognition sites and in the absence of AdoMet. These findings provide evidence that the enzyme actively initiates base flipping by stabilizing the transition state of the process. Reduced rates of base flipping in substrates containing the target base in a non-canonical sequence demonstrate that DNA recognition by the MTase starts before base flipping. DNA recognition, cofactor binding and base flipping are correlated and efficient base flipping takes place only if the enzyme has bound to a cognate target site and AdoMet is available.
School of Engineering and Science, International University Bremen, Campus Ring 1, 28759 Bremen, Germany.
Kirsten
K
Liebert
Andrea
A
Hermann
Martina
M
Schlickenrieder
Albert
A
Jeltsch
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics*
D004269:DNA, Bacterial; D012436:S-Adenosylmethionine; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)