The Deoxyribonucleases of Escherichia coli

Exonuclease I from Escherichiu coli (I) (previously designated as E. coZi phosphodiesterase (2)), shows a high degree of selectivity for denatured or single stranded deoxyribonucleic acid. This enzyme attacks at the 3’-hydroxyl end of a polydeoxyribonucleotide and liberates 5’-mononucleotides in a stepwise manner until the terminal dinucleotide, which is not attacked. Because of these properties and the low level of endonuclease activity in even relatively impure preparations, it has come into widespread use in studies of DNA and polydeoxyribonucleotide structure (3-6). We have purified exonuclease I1 to a stage approximately lo-fold beyond that reported earlier in order to determine whether a more purified enzyme might be a correspondingly more sensitive reagent for discriminating between native and denatured DNA. This has, in fact, proved to be the case. Another aim of this investigation was to examine further the specificity of exonuclease I with the use of oligonucleotides of defined structure and sequence. More specifically, we wished to establish whether attack by this enzyme began exclusively at the 3’-hydroxyl end of a deoxyribo-oligonucleotide or whether attack beginning at the opposite end of the chain (5’.phosphoryl or 5’-hydroxyl end) occurred as well. In addition, we wished to examine the effect of substitution of the 3’-hydroxyl group of an oligonucleotide on the rate at which it was attacked by exonuclease I. These studies have revealed that exonuclease I attacks only at the 3’-hydroxyl end of a deoxyribo-oligonucleotide and that a free 3’-hydroxyl group is essential for enzymatic activity.