THE DEPENDENCE OF DNASE-I ACTIVITY ON THE CONFORMATION OF OLIGODEOXYNUCLEOTIDES

We have developed a sensitive continuous assay for nucleases using pro ton release. The assay has been applied to the determination of the ki netics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of k(cat)/K-m on sequence and structure of short oligonucle otide substrates has been measured: increasing lengths of A(n)T(n) seq uences decrease the rate of cleavage. G . A mismatches in which the ba ses pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G . A mismatches which use amino pairing and have B-II phosphodiesters, are refractory to DNase I. Also, the DNA strand s of DNA RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove- binding ligand propamidine. A value of 3 mu M has been determined for binding to the sequence d(CGCGAATTCGCG)(2), showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.