PMID: 8464886Apr 1, 1993Paper

Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis

Proceedings of the National Academy of Sciences of the United States of America
L Li, S Chandrasegaran

Abstract

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. Recently, we reported the presence of two distinct and separable protein domains within this enzyme--one for the sequence-specific recognition and the other for endonuclease activity. Here, we report the construction of two insertion mutants of Fok I endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, they cleave one nucleotide further away from the recognition site on both strands of the DNA substrates. Thus, it is possible to alter the cleavage distance of Fok I by protein engineering.

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Citations

Oct 18, 1994·Biochimica Et Biophysica Acta·A Yonezawa, Y Sugiura
Mar 31, 1998·Current Opinion in Structural Biology·A K Aggarwal, D A Wah
Feb 1, 1994·Proceedings of the National Academy of Sciences of the United States of America·Y G Kim, S Chandrasegaran
Feb 6, 1996·Proceedings of the National Academy of Sciences of the United States of America·Y G KimS Chandrasegaran
Sep 2, 1998·Proceedings of the National Academy of Sciences of the United States of America·J BitinaiteI Schildkraut
Dec 24, 1998·Nucleic Acids Research·J SmithS Chandrasegaran
Oct 28, 2005·Nucleic Acids Research·Sundar DuraiSrinivasan Chandrasegaran
Mar 24, 2009·Nucleic Acids Research·Shaun M LippowDasa Lipovsek
Oct 24, 2007·Protein Engineering, Design & Selection : PEDS·Penghua ZhangShuang-yong Xu
Sep 24, 1999·Biological Chemistry·S Chandrasegaran, J Smith
Sep 2, 1998·Proceedings of the National Academy of Sciences of the United States of America·D A WahA K Aggarwal
Dec 16, 1997·Proceedings of the National Academy of Sciences of the United States of America·Y G KimA Rich
Jun 1, 2014·Nucleic Acids Research·Alfred PingoudWolfgang Wende
Mar 19, 2014·Nucleus·Stephanie FanucchiMusa M Mhlanga
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May 26, 2016·EcoSal Plus·Kyeong Rok ChoiSang Yup Lee
Jul 27, 2005·Biochemical and Biophysical Research Communications·Mala ManiSrinivasan Chandrasegaran
Nov 26, 2010·Journal of Molecular Biology·Sivaprakash RamalingamSrinivasan Chandrasegaran

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