Journal
NUCLEIC ACIDS RESEARCH
Volume 35, Issue 1, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkl916
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Funding
- NATIONAL CANCER INSTITUTE [P01CA065930] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R15GM067744] Funding Source: NIH RePORTER
- NCI NIH HHS [P01 CA65930-08, P01 CA065930] Funding Source: Medline
- NIGMS NIH HHS [R15 GM067744, GM 067744] Funding Source: Medline
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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.
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