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The integrity of the genetic information encoded by DNA is essential to all living organisms, yet the reactive bases of DNA are constantly attacked by endogenous and exogenous agents resulting in as many as one million individual molecular lesions per cell per day. Excessive DNA damage or deficiency in DNA repair enzymes may cause cancer, premature aging, and neurodegenerative diseases. Endonuclease V (Endo V) is a DNA repair enzyme which can recognize all four types of DNA deamination products, specifically, uracil, hypoxanthine, xanthine and oxanine. It was also shown that endo V can recognize mismatches. We screened about 60 mutants of endo V from Thermotoga maritima and found some mutants had altered base preferences for mismatches. Tma endo V Y80A was shown to become a C-specific mismatch endonuclease. G13D mutation in K-ras oncogene which was not recognized by wild type Tma endo V was successfully cleaved by Tma endo V Y80A. This study provides valuable information on base recognition and active site organization of Tma endo V. Tma endo V mutants can be used for cancer mutation scanning and mutatio