Terminal crosslinking of vaccinia DNA strands by an in vitro system

An in vitro system has been developed to study the formation of crosslinks in vaccinia DNA. Cytoplasmic extracts from virus-infected cells were used as a source of activity to catalyze the crosslinking of DNA molecules. Replicating vaccinia DNA lacking crosslinks was used as substrate for the reaction. Sedimentation analysis, hydroxylapatite chromatography, labeling of the free ends, and digestion with restriction endonucleases showed that these molecules were not crosslinked. The conditions of the crosslinking reaction were similar to those required for the activities of the DNA polymerase and polynucleotide ligase in vaccinia-infected cells. The product of the reaction was analyzed by sedimentation in alkaline sucrose gradients and by hydroxylapatite chromatography. The results indicated that cytoplasmic extracts from virus-infected cells were able to catalyze the production of DNA molecules sedimenting as the 90 and 106 S species. These molecules upon denaturation "snap back" to a double-stranded conformation as demonstrated by hydroxylapatite chromatography. Removal from the incubation mixture of ATP, Mg²⁺, or deoxynucleotides, as well as heating the extract at 100°, prevented the development of crosslinked molecules. When vaccinia DNA labeled at the 3′ or 5′ termini was used as substrate for the reaction, it was shown that loss of radioactivity occurred only at the 3′ termini. Furthermore, after the reaction 5′-³²P-labeled DNA became resistant to alkaline phosphatase implying that the termini were no longer free. Exonuclease, DNA polymerase, and DNA ligase activities were detected in the cytoplasmic extracts of virus-infected cells, suggesting that they may be involved in the crosslinking reaction. Based on the experimental evidence, a model for the formation of crosslinks is proposed.