Recognition of model DNA replication forks by the SV40 large tumor antigen

The ability of the SV40 large tumor antigen (T antigen), a DNA helicase, to bind to model DNA replication forks was tested. DNA fork molecules were constructed either from two partially complementary oligonucleotides or from a single oligonucleotide able to form a ‘panhandle’ structure. T antigen specifically recognized the two-strand fork in a reaction dependent on the presence of ATP, dATP, or non-hydrolyzable analogs of ATP. T antigen asymmetrically bound the two-strand fork, protecting from nuclease cleavage a fork-proximal region on only one of the two strands. The asymmetric binding is consistent with the 3′?5′ directionality of the DNA helicase activity of T antigen. An analogous region on the one-strand fork was also bound by T antigen, suggesting that T antigen does not require a free singlestranded end to load onto the fork. Use of chemically modified DNA substrates indicated that T antigen binding to the fork utilized important contacts with the DNA sugar-phosphate backbone.