期刊
EMBO JOURNAL
卷 26, 期 16, 页码 3815-3825出版社
WILEY
DOI: 10.1038/sj.emboj.7601807
关键词
atomic force microscopy; DNA looping; DNA translocation; restriction enzyme; single molecule
资金
- Biotechnology and Biological Sciences Research Council [BBS/B/1065X] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BBS/B/1065X] Funding Source: Medline
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.
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