331 Biochimica et Biophysica Acta, 653 (1981) 331--343 Elsevier/North-Holland Biomedical Press BBA 99851 TWO CLASSES OF REPLICATING MOLECULES OF ADENOVIRUS TYPE 2 DNA LEDA H. RAPrIS and PIERRE BOURGAUX Departement de Microbiologie, Facultg de Mgdecine, Centre Hospitalier Universitaire, Universitd de Sherbrooke, Sherbrooke, Qudbec JIH 5N4 (Canada) (Received December 19th, 1980) Key words: DNA replication; Ribonuclease H; Klenow enzyme; DNA polymerase I; (Adenovirus 2) Summary Replicating DNA of human adenovirus type 2, identified as partly single- stranded viral DNA in which [3H]thymidine is readily incorporated, was found to be separated into two fractions by chromatography on hydroxyapatite. Whereas one of these fractions was eluted with 180 mM phosphate, the other one was eluted at the same concentration, 240 mM, as fully double-stranded DNA. The physical properties of the 180 and 240 mM fractions, in particular their buoyant densities in solutions of CsC1 and Cs2SO4, were compared, both before and after treatment by various enzymes such as Neurospora crassa nuclease, pancreatic ribonuclease, ribonuclease H and the Klenow fragment of DNA polymerase I of Escherichia coli, used alone or in various combinations. Unlike the 240 mM fraction, the 180 mM fraction was found to include a sub- stantial amount of single-stranded DNA, some of it being hydrogen-bonded to RNA. Both of these features confer to the 180 mM fraction the high buoyant density in cesium salt solution which was described, for several adenoviruses, as one of the characteristic properties of replicating DNA. Introduction The DNA of human adenoviruses is a linear double-stranded molecule with a molecular weight of {20--25) • 106 [1], the replicative forms of which have been first detected in pulse and/or pulse-chase experiments [2--4]. As com- pared with mature viral DNA, replicating adenovirus DNA sediments more rapidly in neutral sucrose solution and is retained preferentially by benzoyl- ated-naphthoylated DEAE-cellulose columns [5]. Such properties denote the 0005-2787/81/0000--0000/$02.50 @) Elsevier/North-Holland Biomedical Press