| Abstract: | Hydroxyapatite chromatography and isopycnic Cs2SO4 centrifugation normally yield no indications of single-stranded DNA when that fraction of replicating DNA from Ehrlich ascites cells which can be separated by nitrocellulose chromatography is analyzed. Single-stranded DNA is detected by both methods if the DNA is fragmented by ultrasound before the nitrocellulose chromatography. The digestion of this DNA fraction by single-strand-specific nucliase leads to a loss of its binding to nitrocellulose and of the indications of single-stranded DNA. The loss for the affinity to nitrocellulose is also observed when the corresponding fraction separated from unfragmented DNA is digested by endonuclease. It is suggested that replicating DNA is bound to nitrocellulose by means of single-stranded gaps on the replication fork. These gaps are apparently too small to be detected within large, otherwise entirely double-stranded molecules by hydroxyapatite chromatography and Cs2SO4 centrifugation. In the case of nitrocellulose-binding ultrasound fragments, this relation seems to be more favorable because of the separation of most of the residual double-stranded part. It is demonstrated that sonication of helical DNA also generates a small amount of fragments with some single-stranded character. The effects observed with replicating DNA could be distinguished from these artifacts. |
