Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase.
The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.
Su
T-J
TJ
School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK.
Connolly
B A
BA
Darlington
C
C
Mallin
R
R
Dryden
D T F
DT
D015075:2-Aminopurine / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015075:2-Aminopurine; D012436:S-Adenosylmethionine; D004247:DNA; C093244:DNA modification methylase EcoKI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)