Size of native and denatured DNA of Ehrlich ascites tumour cells isolated in the presence of different protease concentrations.

Native DNA molecules isolated either in the presence of 50 micrograms x ml(-1) of proteinase K (PK-DNA) or in the presence of 6 mg x ml(-1) autodigested pronase (PRO-DNA) are about equal in size. Since shear forces were avoided as far as possible during the isolation procedure, the largest molecules found were longer than 100 microns. The average length of the traced molecules was 34.2 microns for PK-DNA and 29.7 microns for PRO-DNA. In contrast to PK-DNA the length of PRO-DNA molecules undergoes a dramatic change during denaturation. The average contour length of a denatured PRO-DNA molecules is only 6.9 microns. This reduction in length cannot be explained by shrinkage due to changes in ionic strength, pH and the effect of denaturing agents. Moreover, PK-DNA identically denatured was not dramatically changed in size. From this it must be concluded that PRO-DNA contains more internal ends than PK-DNA. This conclusion is supported by the results indicating that PRO-DNA is much more sensitive to nuclease S1 than PK-DNA. The results are consistent with previously published biochemical data suggesting that chromosomal DNA is 'nicked' or 'gapped' in a protease-catalyzed reaction at distinct protease-sensitive sites.