Covalently closed circles of adenovirus 5 DNA

The genome of adenoviruses is a double-stranded linear DNA molecule with inverted terminal repeats about 100 base pairs (bp) in length1,2 and a terminal protein covalently linked to the 5′ nucleotide of each strand3,4. Both of these features permit the formation of DNA circles, the inverted repeats allowing the circularization of single-stranded DNA5,6 and the terminal protein the joining of one or more molecules to yield double-stranded circles or concatemers7. However, although the existence of covalently closed circles has been postulated7, double-stranded viral DNA purified from virions or infected cells by conventional methods (that is, using proteases and phenol or chloroform) has always been obtained in a linear form8. Here we present evidence for the existence in adenovirus 5 (Ad5) infected cells of novel structures resulting from covalent head-to-tail joining of viral DNA molecules and show that these structures are due at least in part to the formation of covalently closed circles.