Internucleotide protein linkers in Ehrlich ascites cell DNA.
DNA from Ehrlich ascites tumor cells is nicked or gapped by a reaction which is induced by proteases such as autodigested pronase, proteinase K, trypsin, chymotrypsin and subtilisin. The cleavage of the protease-sensitive sites is inhibited by protease inhibitors. The nicks or gaps induced by proteases can be demonstrated by nuclease S1 sensitivity of native DNA and by a change of the sedimentation rate of alkali-denatured DNA. The limit size of denatured DNA released after optimal protease treatment is 8.5 x 10(6) daltons (27 kilo bases). The molecular weight of the native DNA pieces released after nuclease S1 degradation of DNA containing the protease-induced nicks or gaps is in the same order indicating that the protease-sensitive sites are alternatively arranged on the opposite DNA strands at an average distance of 13.5 kilo base pairs. Since the protease-induced nicks or gaps in phosphatase-treated DNA are not attacked by Escherichia coli polymerase I, one or both ends liberated by the protease treatment must be blocked by a material other than phosphate groups. The results are most compatible with peptide/protein linkers joining adjacent single-strand DNA subunits. Alternative explanations such as alkali-stable RNA linkers, protein-protected RNA linkers, site-specific nuclease contaminations in the protease preparations or cellular nucleases activated by the protease treatment are eliminated by the results presented in this paper.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000032:analysis*; D004720:Endonucleases; D019286:Endopeptidase K; D010450:Endopeptidases; D009691:Nucleic Acid Denaturation; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011402:Pronase; D011506:Proteins / Q000032:analysis*; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004273:DNA, Neoplasm; D011506:Proteins; D010664:Phenylmethylsulfonyl Fluoride; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010450:Endopeptidases; D019286:Endopeptidase K; D011402:Pronase