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

TitleSwitching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations.
Publication TypeJournal Article
Year of Publication2007
AuthorsGao H, Huang J, Barany F, Cao W
JournalNucleic Acids Res
Volume35
Issue1
Paginatione2
Date Published2007
ISSN1362-4962
KeywordsAmino Acid Substitution, Base Pair Mismatch, Deoxyribonuclease (Pyrimidine Dimer), DNA Mutational Analysis, Genes, ras, HT29 Cells, Humans, Point Mutation, Substrate Specificity
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.

DOI10.1093/nar/gkl916
Alternate JournalNucleic Acids Res.
PubMed ID17130153
PubMed Central IDPMC1702505
Grant ListGM 067744 / GM / NIGMS NIH HHS / United States
P01 CA65930-08 / CA / NCI NIH HHS / United States

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