The isolation of strand-specific nicking endonucleases from a randomized SapI expression library

Nucleic Acids Research
James C SamuelsonShuang-Yong Xu

Abstract

The Type IIS restriction endonuclease SapI recognizes the DNA sequence 5'-GCTCTTC-3' (top strand by convention) and cleaves downstream (N1/N4) indicating top- and bottom-strand spacing, respectively. The asymmetric nature of DNA recognition presented the possibility that one, if not two, nicking variants might be created from SapI. To explore this possibility, two parallel selection procedures were designed to isolate either top-strand nicking or bottom-strand nicking variants from a randomly mutated SapI expression library. These procedures take advantage of a SapI substrate site designed into the expression plasmid, which allows for in vitro selection of plasmid clones possessing a site-specific and strand-specific nick. A procedure designed to isolate bottom-strand nicking enzymes yielded Nb.SapI-1 containing a critical R420I substitution near the end of the protein. The top-strand procedure yielded several SapI variants with a distinct preference for top-strand cleavage. Mutations present within the selected clones were segregated to confirm a top-strand nicking phenotype for single variants Q240R, E250K, G271R or K273R. The nature of the amino acid substitutions found in the selected variants provides evidence that SapI ma...Continue Reading

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Citations

Feb 4, 2006·Nucleic Acids Research·James C SamuelsonShuang-yong Xu
Jun 15, 2007·Nucleic Acids Research·Yu Zheng, Richard J Roberts
Sep 11, 2007·Nucleic Acids Research·Traci KieslingMark Danielsen
Dec 4, 2009·Nucleic Acids Research·Priscilla Hiu-Mei TooShuang-yong Xu
Nov 13, 2010·Nucleic Acids Research·Nicholas LuzziettiRalf Seidel
Mar 3, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Galina S KachalovaHans D Bartunik
Mar 10, 2010·Biochemistry. Biokhimii︠a︡·L A ZheleznayaN I Matvienko
Mar 12, 2009·Proceedings of the National Academy of Sciences of the United States of America·Audrey McConnell SmithBarry L Stoddard
Feb 24, 2011·Analytical Biochemistry·Aric Joneja, Xiaohua Huang
Sep 15, 2009·Protein Expression and Purification·Penghua ZhangShuang-yong Xu
May 15, 2007·Journal of Molecular Biology·Arturas JakubauskasArvydas Janulaitis
Jun 2, 2006·Protein Expression and Purification·Siu-hong ChanShuang-yong Xu
Apr 14, 2005·Journal of Molecular Biology·Daniel F HeiterGeoffrey G Wilson
May 25, 2016·Biochimica Et Biophysica Acta·Liudmila A AbrosimovaTatiana S Oretskaya
Mar 21, 2017·Extremophiles : Life Under Extreme Conditions·Ravinder KumarBeena Krishnan
Dec 27, 2016·Biology Methods and Protocols·Ilya B Tikh, James C Samuelson
Dec 4, 2020·Nucleic Acids Research·Alexey FomenkovRichard J Roberts
Oct 7, 2021·Chemical Society Reviews·Yanan TangX Chris Le

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