Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations.
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.
Gao
Honghai
H
Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, Room 219, Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634, USA.
Huang
Jianmin
J
Barany
Francis
F
Cao
Weiguo
W
D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics* / Q000378:metabolism*; D011905:Genes, ras; D019073:HT29 Cells; D006801:Humans; D017354:Point Mutation; D013379:Substrate Specificity
D043224:Deoxyribonuclease (Pyrimidine Dimer)