Evidence for blocked 5′-termini in human adenovirus DNA

Abstract

DNA molecules from six human adenovirus serotypes, representative of three oncogenic classes, were investigated using enzymes specific for 5′-, and for 3′-termini in DNA. Results obtained with DNA purified by sequential treatment with papain, sodium dodecyl sulfate, and phenol show that the majority of the 5′-termini of all these DNAs is blocked. Depending upon the source of DNA, from 50 to 80% of the 5′-termini are inaccessible to polynucleotide kinase even after extensive alkaline phosphatase treatment. The DNA molecules with 5′-termini inaccessible to phosphorylation are also resistant to λ exonuclease which hydrolyzes DNA starting from 5′-ends. These 5′-termini remain inaccessible to phosphorylation after high temperature phosphatase treatment, or after phosphatase treatment of denatured, single-stranded DNA. However, about 40% of the blocked 5′-termini of adenovirus 2 DNA becomes accessible to phosphatase and polynucleotide kinase after prolonged treatment of the DNA with sodium hydroxide. These findings suggest that a strongly bonded substituent rather than a peculiar DNA conformation accounts for the inaccessible 5′-termini. The 5′-blocking group does not affect the reactivity of the 3′-termini since all of the molecules are attacked by exonuclease III, which hydrolyzes DNA starting from 3′-ends. Finally, in all of the adenovirus serotypes examined, the nucleotide found at the 5′-termini of DNA molecules accessible to labeling is predominantly deoxycytidylate.