Abstract
Abstract 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.
Highlights
Introduction of0.15 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.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, and, the enzyme should be classified as a polynucleotidase.No endonuclense is known with an absolute specificity toward either native or denatured DNA
The reaction rate during the first phase is proportional to Ca++ concentration
Since each has a different requirement for Ca++, the relative hydrolytic rates vary widely with Ca++ concentration
Summary
Introduction of0.15 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.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, the enzyme should be classified as a polynucleotidase.No endonuclense is known with an absolute specificity toward either native or denatured DNA. 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. Micrococcal nuclease hydrolyzes denatured DNA much faster than native DNA. In the presence of both substrates, it does not hydrolyze denatured DNA exclusively. No endonuclense is known with an absolute specificity toward either native or denatured DNA. It may be expected that kinetics of hydrolytic degradation of DNA will be complicated by the effect of conformational change. A nuclease that more readily attacks the double-stranded form will show autoretardation, because the double-stranded structure does not persist for long. The word autoretardation was coined to characterize kinetic changes observed with DNase I (2). Even more pronounced changes of a similar nature have been previously described with DNase II (3)
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