Fluorescence study of DNA binding and bending by EcoRI DNA methyltransferase.
We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .
Ma
Baocheng
B
Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China.
Wang
Jun
J
Fang
Xiaohong
X
D000225:Adenine / Q000737:chemistry; D016880:Anisotropy; D002627:Chemistry, Physical / Q000379:methods; D004247:DNA / Q000737:chemistry*; D019793:Fluorescein / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D008745:Methylation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012235:Rhodamines / Q000737:chemistry*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics
D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine; D019793:Fluorescein