Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.