Exploring The Spatiotemporaloral Dynamics of DNA Binding and Cleavage by Restriction Endonucleaes

Abstract

Using restriction endonucleases to catalyze the double-stranded DNA (dsDNA) breakage at certain recognition sequences is an important molecular biology technique. The restriction endonucleases constitute an important defense mechanism of bacteria against viral attacks; this mechanism is to destroy invading foreign DNA molecules via cleaving a specific site (phosphodiester bond) of a dsDNA. By cleaving recognition sites on dsDNA with extraordinary specificity can lead to the DNA double strand breaks (dsb). We presented a novel single-molecule approach to investigate the interaction between DNA and restriction endonucleases, including DNA recognition and cleavage. To elucidate how fast restriction endonucleases recognize and cleavage DNA sequence, we constructed a high resolution dual-beam laser tweezers system to manipulate single DNA molecule, together with the site-specific restriction enzymes, namely, EcoRI (one-site endonuclease) and Cfr9I (two-site endonuclease), conjugated to nanometer-sized fluorescence particle. Because most endonucleases work in the presence of magnesium ions, we will apply optically based reaction mechanism to control and synchronize the restriction endonuclease activity in this study. Furthermore, both laser tweezers and fluorescence particle imaging will be used to probe whether the DNA double strand breaks occurred due to the molecular cutting. Hence, this single-molecule approach allows us to directly observe and visualize the spatiotemporal dynamics of DNA binding and cleavage by restriction endonucleases, and can be further applied to determine the DNA cleavage rate due to the presence of EcoRI and Cfr9I. Finally, we extend this approach, together with the light-induced molecular cutting, to investigate the DNA binding and cleavage by restriction endonucleases under tension at different temperatures.