Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping.
Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 +/- 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.
Crampton
Neal
N
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom.
Yokokawa
Masatoshi
M
Dryden
David T F
DT
Edwardson
J Michael
JM
Rao
Desirazu N
DN
Takeyasu
Kunio
K
Yoshimura
Shige H
SH
Henderson
Robert M
RM
D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism* / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D013997:Time Factors
D004247:DNA; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific