Mode of action of nuclease P1 on nucleic acids and its specificity for synthetic phosphodiesters.

Abstract

Nuclease P1 was found to attack RNA and heat-denatured DNA in endo- and exonucleolytic manners. The evidence was as follows: (1) In the early stage of digestion both mononucleotides and oligonucleotides with various sizes were formed simultaneously with rapid fragmentation of polynucleotides. (2) The relative amount of the monomer was larger than that of any class of oligomers throughout the process of digestion. Nuclease P1 showed a preference for the linkages between 3′-hydroxyl group of adenosine or deoxyadenosine and the 5′-phosphoryl group of the adjacent nucleotides. p-Nitrophenyl ester of 3′-dTMP was hydrolyzed to thymidine and p-nitrophenyl phosphate, while p-nitrophenyl ester of 5′-dTMP was not attacked. It is concluded from these findings that the basic structure required for the substrate of nuclease P1 is a nucleoside 3′-phosphate-containing structure and the enzyme cleaves the diester bond between the phosphate and the 3′-hydroxyl group of the sugar.