Reddy, Yeturu VR and Rao, Desirazu N (2000) Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence. In: Journal of Molecular Biology, 298 (4). pp. 597-610.
Restricted to Registered users only
Download (368Kb) | Request a copy
EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5′-CAGCAG-3′ transferring a methyl group from S-adenosyl-$L$-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Academic Press|
|Keywords:||base flipping;DNA distortion;adenine methyltransferase;DNA-protein interactions|
|Department/Centre:||Division of Biological Sciences > Biochemistry|
|Date Deposited:||17 Oct 2007|
|Last Modified:||19 Sep 2010 04:40|
Actions (login required)