Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen.

The mechanism by which DNA helicases unwind DNA was tested; an "unwinding complex" between the SV40 large tumor antigen (T antigen) and a DNA molecule designed to resemble a replication fork was probed. In an adenosine triphosphate (ATP)-dependent reaction, T antigen quantitatively recognized this synthetic replication fork and bound the DNA primarily as a hexamer. The T antigen bound only one of the two strands at the fork, an asymmetric interaction consistent with the 3'----5' directionality of the DNA helicase activity of T antigen. Binding to chemically modified DNA substrates indicated that the DNA helicase recognized the DNA primarily through the sugar-phosphate backbone. Ethylation of six top strand phosphates at the junction of single-stranded and double-stranded DNA inhibited the DNA helicase activity of T antigen. Neither a 3' single-stranded end on the DNA substrate nor ATP hydrolysis was required for T antigen to bind the replication fork. These data suggest that T antigen can directly bind the replication fork through recognition of a fork-specific structure.