As a rule, when restriction endonucleases bind to their recognition sequences in DNA, they hydrolyze both strands of the duplex at the same time. Two independent hydrolytic reactions proceed in parallel, most often driven by the presence of two catalytic sites within each enzyme, one for hydrolyzing each strand. We have begun to engineer altered restriction enzymes that hydrolyze only one strand of the duplex, to produce DNA molecules that are “nicked”, rather than cleaved. These conventional nicks (3´-hydroxyl, 5´-phosphate) can serve as initiation points for a variety of further enzymatic reactions such as replacement DNA synthesis, strand-displacement amplification (1), exonucleolytic degradation or the creation of small gaps (2).
(1) Walker, G.T. et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 392–396. PMID: 1309614
(2) Wang, H. and Hays, J.B. (2000) Mol. Biotechnol., 15, 97–104. PMID: 10949822
(3) Higgins, L.S. et al. (2001) Nucleic Acids Res., 29, 2492–2501. PMID: 11410656
(4) Morgan, R.D. et al. (2000) Biol. Chem., 381, 1123–1125. PMID: 11154070
(5) Zhu, Z. and Xu, S.Y. unpublished results.
(6) Xu, Y. et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 12990–12995. PMID: 11687651
(7) Heiter, D.F. et al. (2005) J. Mol. Biol., 348, 631–40. PMID: 15826660
(8) Samuelson, J.C., Zhu, Z. and Xu, S.Y. (2004) Nucleic Acids Res., 32, 3661–3671. PMID: 15247348
(9) Zhu, Z. et al. (2004) J. Mol. Biol., 337, 573–583. PMID: 15019778
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Watch as Geoff Wilson, Restriction Enzyme Division Head, describes the interaction of restriction enzymes and substrate DNA using computer models generated from x-ray crystallography data.
Watch as Rick Morgan, Research Scientist in the Restriction Enzyme Division, describes his passion for discovering and characterizing restriction enzymes from nature.