The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence...

Using a series of decadeoxyribonucleotides containing base analogues as substrates we measured the steady-state kinetic parameters for the reaction catalyzed by RsrI endonuclease and compared the results to those with its isoschizomer EcoRI. The kinetics of RsrI cleavage are affected by each substitution, with the effects being generally more deleterious than with EcoRI, as shown by the greater reduction in the specificity constant kcat/KM. The magnitudes of the effects of several substitutions are consistent with the formation of direct enzyme-nucleobase contacts at the indicated positions. With substrates containing 2-amino-purine or 2,6-diaminopurine at the central adenine or uracil at the outermost thymine in the recognition sequence, cleavage by RsrI was very slow, less than one-tenth the rate of the corresponding EcoRI-catalyzed reaction. The lower tolerance of RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms of DNA recognition by the two enzymes. Although RsrI and EcoRI endonucleases bind with similar affinities to specific and nonspecific DNA sequences and appear to introduce similar structural distortions in DNA upon binding, the use of substrate analogues reveals significant differences at the level of catalysis in the mechanisms by which these two endonucleases recognize the duplex sequence GAATTC.