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.
Crampton
Neal
N
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK.
Roes
Stefanie
S
Dryden
David T F
DT
Rao
Desirazu N
DN
Edwardson
J Michael
JM
Henderson
Robert M
RM
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