MISMATCH-SPECIFIC DNA BREAKDOWN IN NUCLEAR EXTRACT FROM TOBACCO (NICOTIANA-TABACUM) CALLUS

Mismatch-specific enzymatic activity was sought for in nuclei from normal and transformed plant cells originating from tobacco (Nicotiana tabacum) callus and crown gall tumor induced by Agrobacterium tumefaciens. The specific enzymatic activity was assayed with substrates derived from synthetic oligonucleotides (19-mer sequences corresponding to the human K-ras gene). Single-base changes in the middle of the sequence were the basis for creating heteroduplexes with all eight mismatches. Homo- and heteroduplexes were ligated in a size ladder and used as substrates. We detected mismatch-specific DNA breakdown and determined basic requirements for the reactions. Kinetic analysis indicates the following reactivity order of preference:C:A = C:C = C:T > G:T approximately A:A approximately G:A approximately G:G approximately T:T > > G:C. It can be said now that specific mismatch recognition and repair activities have been detected in all kingdoms of living species.