Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands
Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting 'top' and 'bottom' strands 9 and 13 nucleotides downstream of the...
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| Veröffentlicht in: | Nucleic acids research 2009-04, Vol.37 (7), p.2105-2115 |
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| creator | Sanders, Kelly L Catto, Lucy E Bellamy, Stuart R.W Halford, Stephen E |
| description | Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting 'top' and 'bottom' strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting. |
| doi_str_mv | 10.1093/nar/gkp046 |
| format | Article |
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| subjects | Deoxyribonucleases, Type II Site-Specific - genetics Deoxyribonucleases, Type II Site-Specific - metabolism DNA - chemistry DNA - metabolism Gene Targeting Kinetics Nucleic Acid Enzymes Protein Subunits - genetics Protein Subunits - metabolism Substrate Specificity |
| title | Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands |
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