The Occurrence of Autoacceleration during Hydrolysis of Nucleic Acids by Micrococcal Nuclease

In the course of digestion of nucleic acids by micrococcal nuclease in the presence of low Ca++ concentration, three distinct phases of the reaction are observed. The first and the third are slow, and the second is fast. The reaction rate during the first phase is proportional to Ca++ concentration. An autoaccelerated second phase, showing little dependence on Ca++ concentration, has been observed with native DNA, denatured DNA, and RNA. The apparent end point (termination of the third phase) occurs earlier with decreasing Ca++ concentration. Of the three substrates studied, native DNA, RNA, and denatured DNA, native DNA is the most resistant. However, since each has a different requirement for Ca++, the relative hydrolytic rates vary widely with Ca++ concentration. Introduction of 0.15 m NaCl into the reaction medium accelerates the first phase and decelerates the second phase. The over-all reaction is slower, and the extent of hydrolysis is decreased. Under identical conditions, micrococcal nuclease hydrolyzes denatured DNA much faster than native DNA. However, in the presence of both substrates, it does not hydrolyze denatured DNA exclusively. The autoacceleration phenomenon suggests that the best substrate for micrococcal nuclease is a polynucleotide chain of intermediate length (between nucleic acids and short oligonucleotides), and, therefore, the enzyme should be classified as a polynucleotidase.