Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region.
Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rate of 1-2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.
Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Yuki
Y
Suzuki
Jamie L
JL
Gilmore
Shige H
SH
Yoshimura
Robert M
RM
Henderson
Yuri L
YL
Lyubchenko
Kunio
K
Takeyasu
D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000648:ultrastructure; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific