The role of simian virus 40 (SV40) large tumor antigen (T antigen) as a DNA helicase at the replication fork was studied. We found that a T-antigen hexamer complex acts during the unidirectional unwinding of appropriate DNA substrates and is localized directly in the center of the fork, contacting the adjacent double strand as well as the emerging single strands. When bidirectional DNA unwinding, initiated at the viral origin of DNA replication, was analyzed, a larger T-antigen complex that is simultaneously active at both branch points of an unwinding bubble was observed. The size and shape of this helicase complex imply that the T-antigen dodecamer complex, assembled at the origin and active in the localized melting of duplex DNA, is subsequently also used to continue DNA unwinding bidirectionally. Then, however, the dodecamer complex does not split into two hexamer subunits that track along the DNA; rather, the DNA is threaded through the intact complex, with the concomitant extrusion of single-stranded loops.

DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5′ nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid‐point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT‐nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5′ terminal nucleotide. Deletion in the amino‐terminal part of the traI polypeptide abolishes the oriT‐nicking activity while leaving the strand‐separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site‐specific endonuclease and strand‐separating activities.

Simian virus 40 (SV40) large tumor antigen (T antigen) is mainly localized in the nucleus where it exhibits two biochemical properties: DNA binding and helicase activity. Both activities are necessary for viral DNA replication and may also enable T antigen to modulate cellular growth. Here we present biochemical and electron microscopic evidence that the helicase activity can start at internal sites of fully double-stranded DNA molecules not containing the SV40 origin or replication. Using T antigen specific monoclonal antibodies, this unwinding reaction can be biochemically divided in an initiation (duplex opening) and a propagation step. The duplex opening reaction (as well as the propagation step) does not depend on a specific DNA sequence or secondary structure. In addition, we have found that T antigen forms an ATP dependent nucleoprotein complex at double-stranded DNA, which may be an essential step for the sequence independent duplex DNA opening reaction.

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Simian virus 40 large tumour antigen (T antigen) is shown to catalyse the formation of duplex DNA from complementary strands in specific conditions. The activity is dependent on an excess of unspecific double‐stranded DNA and seems not to function by T antigen mediated destabilization of secondary structure. Rather, protein‐protein contacts between T antigen molecules appear to be involved. Protein‐protein interactions between T antigen molecules bound to physically separated DNA sites are also demonstrated by the formation of specific DNA loops and by cyclization of DNA molecules with 3′‐extended single‐stranded ends where T antigen specifically binds to the single‐stranded/double‐stranded junctions. The relevance of these properties for T antigen functions in DNA replication, transcription and(or) recombination is discussed.