The TaqI star reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition

Abstract Taq I endonuclease recognizes and cleaves its canonical sequence, TCGA, with complete fidelity under standard conditions. In the presence of some organic solvents, Taq I endonuclease introduced additional single-strand and double-strand cuts at sequences termed Taq I ‘star’ sites. Using ‘middle-labeled’ DNA, the relative rates of cleavage of each strand were simultaneously determined for several star sites. These star recognition sequences differed from the canonical sequence by a single base, and all potential star sites were either nicked or cleaved. Star sites within the middle labeled substrate represented ten of the twelve possible star sequences for each strand. For each group of identical star sites, one strand was consistently preferred for cleavage. Based on these preferences, a model for Taq I recognition of the TCGA sequence is proposed. According to this model, sequence discrimination is mediated by eight hydrogen bonds formed between Taq I and the cognate nucleotides within the major groove.