Initiation and termination sites of adenovirus 12 DNA replication II. Analysis of pulse-labeled oligonucleotides derived from 5′ termini in the DNA strand

Abstract

The initiation site of adenovirus type 12 (Ad12) DNA replication was investigated by using a temperature-sensitive (ts) mutant, defective in initiation of viral DNA replication at the restrictive temperature. Ad12 DNA pulse labeled with [ 3H]thymidine at the initiation site was mixed with U- 32P-labeled Ad12 DNA and cleaved by endonuclease at varying concentrations to produce oligonucleotides with different average lengths. The oligonucleotides were further digested with an appropriate exonuclease to leave the 5′-end oligonucleotides intact but digest the other oligonucleotides completely, or to digest the 5′-end oligonucleotides selectively but leave the other oligonucleotides intact. The oligonucleotides were separated from the mononucleotides by either gel filtration or DEAE-Sephadex column chromatography. Comparison of the elution profiles of the 3H and 32P label revealed that the 3H label could not be incorporated into small 5′-end oligonucleotides produced by a complete digestion with spleen DNase II, whereas a significant amount of 32P label was always recovered in these small oligonucleotides. These results suggest that the replication of Ad12 starts at a short distance from the 5′ end of the strand, but does not start right at the 5′ end. Contrary to the initiation site, the results suggest that the replication of Ad12 DNA terminates probably right at the 3′ end of the strand.