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Nucleic Acids Research Advance Access published online on November 27, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl916
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© 2006 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.

Methods Online

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Honghai Gao, Jianmin Huang1, Francis Barany1 and Weiguo Cao*

Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, Room 219 Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634, USA 1 Department of Microbiology and Immunology, The Joan and Sanford I. Weill Medical College of Cornell University 1300 York Avenue Box 62, NY 10021, USA

*To whom correspondence should be addressed. Tel: +1 864 656 4176; Fax: +1 864 656 0393; Email: wgc{at}clemson.edu

Received August 15, 2006. Revised October 5, 2006. Accepted October 16, 2006.

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3' to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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