Mismatch Endonuclease I prefers to cleave T:T, G:G and T:G DNA mismatches, but will also readily cleave T:I, G:I and G:U DNA mismatches. Additionally, Mismatch Endonuclease I will nick the thymine containing strand of a T:C DNA mismatch, and cleave T:G and T:U (DNA:RNA) mismatches albeit to a lesser extent than DNA:DNA mismatches.