Mode of Action of Acid Deoxyribonucleases from Human Gastric Mucosa and Cervix Uteri

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.