DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes.
EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK.
Neal
N
Crampton
Stefanie
S
Roes
David T F
DT
Dryden
Desirazu N
DN
Rao
J Michael
JM
Edwardson
Robert M
RM
Henderson
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015263:Deoxyribonucleases, Type III Site-Specific / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D012097:Repressor Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; C093225:DNA restriction enzyme EcoPI; D015263:Deoxyribonucleases, Type III Site-Specific