Abstract
The modes of endonucleolytic action of acid deoxyribonucleases (DNases) purified from human gastric mucosa and cervix uteri were investigated. The sedimentation patterns in alkaline and neutral sucrose gradients of digests of linear duplex DNA at an early stage of hydrolysis suggested that gastric DNase makes double strand scissions, while the uterine enzyme makes single strand scissions. To examine the initial kinetics of hydrolysis of the substrate molecule in more detail, λdv twisted circular duplex DNA was used as substrate, and the hydrolysis products were treated with ATP-dependent DNase, which acts specifically on double-stranded DNA in the linear form. The resulting products were sequentially analyzed by sucrose density gradient sedimentation. In this way open circular DNA and linear DNA formed from twisted, circular duplex DNA could be distinguished clearly. Using this method it was found that gastric DNase produces both open circular DNA and linear DNA simultaneously at a very early stage of hydrolysis, while the uterine enzyme produces open circular DNA first, and linear DNA after a lag phase. At 0° the gastric enzyme converted twisted, circular duplex DNA to the open circular form, introducing single strand scissions. These results indicate that both enzymes attack duplex DNA substrate molecules by making single strand scissions, but that the gastric enzyme makes single strand breaks first, followed immediately by second single strand scissions, resulting in the rapid formation of double strand cleavages.
Highlights
To examine the initial kinetics of hydrolysis of the substrate molecule in more detail, Xdv twisted circular duplex DNA
Using this method it was found that gastric DNase produces both open circular DNA
Using sedimentation analysis in conjunction with ATEdependent DNase treatment it was possible to determine the exact proportions of open circular and linear DNA molecules produced on hydrolysis of twisted, circular duplex DNA by the enzymes and the amount of substrate remaining (Fig. 1)
Summary
The modes of endonucleolytic action of acid deoxyribonucleases (DNases) purified from human gastric mucosa and cervix uteri were investigated. The procedure was based on recent reports that ATP-dependent DNase purified from Micrococcus Z&us and other microorganisms hydrolyzes doublestranded DNA in the linear form to acid-soluble oligonucleotides but does not attack circular duplex molecules with or without single strand breaks [12, 13]. Using sedimentation analysis in conjunction with ATEdependent DNase treatment it was possible to determine the exact proportions of open circular and linear DNA molecules produced on hydrolysis of twisted, circular duplex DNA by the enzymes and the amount of substrate remaining (Fig. 1) Results showed that these procedures are useful for analysis of the kinetics of the initial attack of endonucleases on the DNA molecule. Open circles indicate the sedimentation of Escherichia coli [32P]DNA added as a reference of linear DNA, indicating the change in the sedimentation pattern after treatment with ATP-dependent
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