Targeted Base Stacking Disruption by the EcoRI DNA Methyltransferase
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- 作者：Barrett W. Allan and Norbert O. Reich
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：November 26, 1996
- 年：1996
- 卷：35
- 期：47
- 页码：14757 - 14762
- 全文大小：318K
- 年卷期：v.35,no.47(November 26, 1996)
- ISSN：1520-4995
文摘
We describe a novel fluorescence-based assay for detecting DNAconformational alterationswithin enzyme-DNA complexes. The target adenine forEcoRI DNA methyltransferase (GAATTC)wasreplaced with 2-aminopurine, which fluoresces upon excitation at 310nm. Addition of the methyltransferaseto the duplex binding site results in a 14-fold increase influorescence intensity with a 10 nm blue shift.The fluorescence is ~50% of that observed with equimolar freenucleoside, consistent with extrahelicalstabilization of the target base in the enzyme-DNA complex. Theshift in 
max further implies the baseis placed into a low dielectric environment. For adenine-specificDNA methyltransferases, a hydrophobicpocket composed of highly conserved amino acids lies proximal to thecofactor binding site. Substitutionof 2-aminopurine adjacent to the target base also results in detectablechanges in fluorescence emissionfollowing complex formation with the methyltransferase. Thus,other classes of enzymes hypothesizedto utilize base flipping can be investigated by thismethod.
max further implies the baseis placed into a low dielectric environment. For adenine-specificDNA methyltransferases, a hydrophobicpocket composed of highly conserved amino acids lies proximal to thecofactor binding site. Substitutionof 2-aminopurine adjacent to the target base also results in detectablechanges in fluorescence emissionfollowing complex formation with the methyltransferase. Thus,other classes of enzymes hypothesizedto utilize base flipping can be investigated by thismethod.