Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations
Open Access
- 27 November 2006
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 35 (1), e2
- https://doi.org/10.1093/nar/gkl916
Abstract
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.Keywords
This publication has 26 references indexed in Scilit:
- Enzymatic Mutation Detection TechnologiesBioTechniques, 2005
- Plastic Contaminant Masquerades as DNA in Mutation Detection by Denaturing HPLCBioTechniques, 2003
- Chemical cleavage reactions of DNA on solid support: application in mutation detectionBMC Chemical Biology, 2003
- The Use of Resolvases T4 Endonuclease VII and T7 Endonuclease I in Mutation DetectionMolecular Biotechnology, 2003
- An amplification and ligation-based method to scan for unknown mutations in DNAHuman Mutation, 2002
- An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissueOncogene, 2002
- Mutation Detection by Denaturing DNA Chromatography Using Fluorescently Labeled Polymerase Chain Reaction ProductsAnalytical Biochemistry, 1999
- Use of a DNA toolbox for the characterization of mutation scanning methods. I: Construction of the toolbox and evaluation of heteroduplex analysisElectrophoresis, 1999
- Use of DNA toolbox for the characterization of mutation scanning methods. II: Evaluation of single-strand conformation polymorphism analysisElectrophoresis, 1999
- Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvasesNature Genetics, 1995