The DNA-binding domain of simian virus 40 tumor antigen has multiple functions.

The DNA-binding domain of simian virus 40 tumor antigen has been previously shown to participate in a number of different activities. Besides being involved in binding to sequences at the viral replication origin, this domain appears to be required for nonspecific DNA binding, for structurally distorting origin DNA (melting and untwisting), and possibly for oligomerization of the protein into hexamers and double hexamers. We now provide evidence that it also takes part in unwinding origin DNA sequences, contributes a function specifically related to in vivo DNA replication, and perhaps supports the assembly of the virus or release of the virus from the cell.

Biochemical analysis of mutants with changes in the origin-binding domain of simian virus 40 tumor antigen.

The role of the origin-binding domain of simian virus 40 large tumor antigen (T antigen) in the initiation of virus DNA replication was investigated by analyzing the biochemical activities of a series of mutants with single-site substitutions in this region. These activities include origin-specific and nonspecific DNA binding, melting of the imperfect palindromic sequence, untwisting of the AT-rich region, unwinding of origin-containing DNA, helicase activity, and the ability to oligomerize normally in response to ATP. Three classes of T-antigen mutants that are unable to support virus replication in monkey cells are described.

Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity.

We have previously identified three regions (called elements) in the DNA-binding domain of simian virus 40 large tumor (T) antigen which are critical for binding of the protein to the recognition pentanucleotides GAGGC at the viral replication origin. These are elements A (residues 147 to 159), B1 (185 to 187), and B2 (203 to 207). In this study, we generated mutants of simian virus 40 in order to make single-point substitution mutations at nearly every site in these three elements.

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated.