Nucleic Acids Research Advance Access originally published online on March 20, 2009
Nucleic Acids Research 2009 37(9):3061-3073; doi:10.1093/nar/gkp182
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Nucleic Acids Research, 2009, Vol. 37, No. 9 3061-3073
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Creation of a type IIS restriction endonuclease with a long recognition sequence
a LipovekCodon Devices, Inc., 99 Erie Street, Cambridge, MA, 02139, USA
*To whom correspondence should be addressed. Tel: +1 617 871 1174; Fax: +1 617 876 0673; Email: slippow{at}gmail.com
Received February 4, 2009. Revised February 27, 2009. Accepted March 5, 2009.
Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.