A unique form of terminal redundancy has been observed in DNA molecules extracted from several human adenovirus serotypes. Electron microscopic studies reveal that single-stranded circular molecules are formed when native DNA is denatured and then annealed.Temperatures approaching the T. of native DNA are required to convert circles to linear molecules, indicating a high degree of self-complementarity between terminal base sequences of DNA strands. Single-stranded circles are not generated if a limited number of nucleotides (2-4%) are removed from the 3' ends of native DNA by digestion with Escherichia coli exonuclease III before denaturation and annealing. The length of the redundant segment appears to differ among major serotypic groups, and a possible association between increased length of the redundant segment and increased oncogenic capability of virus serotype is suggested. Evidence for the configuration of the duplex closure region of circular molecules is also presented.Human adenovirus DNA is double-stranded and linear, with a molecular weight of 20 to 25 X 106 (1, 2). Because native DNA molecules do not circularize on annealing and form few, if any, circles when annealed after treatment with Escherichia coli exonuclease III, it has been thought that the adenovirus genome does not contain a terminal redundancy (3). However, during studies with DNA from an adenovirus 7-SV40 hybrid virus (4), occasional single-stranded circular molecules were seen in preparations of renatured DNA (T. J. Kelly and J. A. Rose, unpublished observations). A selfannealing of single DNA strands into circular molecules would be evidence for terminal redundancy in adenovirus DNA.In the present report, we show that denatured DNA from several human adenovirus serotypes can be readily selfannealed into single-stranded circular molecules. This finding indicates that adenovirus DNA contains a previously undescribed type of terminal repetition, i.e., the repeated base sequences have exchanged strands, resulting in single-stranded molecules with complementary terminal segments. Based on the extent of exonuclease III digestion required to prevent circle formation, it is estimated that terminal repetitions may represent up to 4% of viral DNA, depending on serotype. MATERIALS AND METHODSViruses. Human adenovirus serotypes 2, 3, 7, and 12 were obtained from W. P. Rowe. Types 1 and 18 were provided by J. C. Hierholzer and type 31 was obtained from H. Shimojo. All are prototype strains except for type 7, which is strain E46-(4). Stock pools were prepared by passage in human embryonic kidney cells (HEM Research, Inc., RockAbbreviation: Ad, adenovirus. ville, Md.). Viruses were produced in KB cells in suspension culture and purified as described (5).Purification and Analysis of Viral DNA. The extraction of intact adenovirus DNA from bands of CsCl-purified virus has been described in detail (4). DNA preparations were stored in a Tris-EDTA buffer [10 mM Trise HC1 (pH 8.5)-i mM EDTA] at 4°. Ad 2 DNA labeled with 82p (6) was unbroken...

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