Kinetics of the Middle and Terminal Phases of Degradation of DNA by Spleen Acid DNAase

Abstract

The kinetics of the middle and terminal phases of calf thymus DNA degradation by spleen acid DNAase has been investigated in detail with the main purpose of providing a background for a study of the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA.During the middle phase the ultraviolet absorption, the acid solubility and the reciprocal average degree of polymerization of the hydrolysate, ¯n−1, increase linearly with digestion time, while the elution profile of the DEAE‐cellulose‐urea chromatogram is progressively shifted to the left. Empirical relationships between ¯n−1 and hyperchromic shift have been established. Extrapolation of ¯n−1 to zero digestion time indicates that bond splitting proceeds at a faster, yet decreasing rate during the early part of digestion.The beginning of the terminal phase is characterized by a sudden slow down of the reaction rate; this phenomenon has been shown to be essentially due to the melting of double‐stranded DNA fragments; the single‐stranded fragments so originated are a poor substrate for the enzyme, in spite of the fact that they still contain a large number of sequences which can be split. During the terminal phase, the ultraviolet absorption, the acid solubility and ¯n−1 increase at a progressively slower rate until an end‐point is practically reached. Investigations on the liberation of mono‐ and dinucleotides, the base composition and termini of isostichs and the effects of digestion temperature and of dephosphorylation on the isostich patterns are also reported.