Quantitative analysis of EcoR1 methylase‐DNA complex by atomic force microscopy

Abstract

The EcoR1 methylase specifically recognize 5'-GA* ATTC-3' in DNA duplex. We directly applied atomic force microscopy (AFM) to investigate linear pBR322-EcoR1 methylase complexes and quantitatively analyzed the bend angles of linear pBR322-EcoR1 methylase complexes and the bound protein widths. In this study, we made a novel observation that DNA-EcoR1 methylase complexes exhibited two populations of conformation at recognition site: DNA bent an acute angle at the recognition site in the presence of one EcoR1 methylase monomeric molecule, while DNA bent an unacute angle at the recognition site and the complementary site on duplex DNAs in the presence of EcoR1 methylase dimer. The data indicated that the unacute angle state was the result of unique interactions between EcoR1 methylase and the recognition site and the complementary site on duplex DNAs, and suggested that the acute angle conformation could be an intermediate in the formation of the unacute angle state. Our works provide a detail insight into the DNA structural variations involved in EcoR1 methylase-binding processes and demonstrate further the versatility of AFM as an imaging technique for studying the interaction between large DNA fragment and protein.