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Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen
1Department of Biochemistry, New York University Medical Center, NY 10016.
Abstract:
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.
Insights
SV40 large tumor antigen (T antigen), a DNA helicase, binds replication forks asymmetrically. It recognizes DNA via the sugar-phosphate backbone, independent of ATP hydrolysis, suggesting a fork-specific binding mechanism.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- DNA helicases are crucial enzymes that unwind DNA.
- The SV40 large tumor antigen (T antigen) possesses DNA helicase activity.
- Understanding the precise mechanism of DNA unwinding by T antigen is essential.
Purpose of the Study:
- To investigate the mechanism of DNA unwinding by SV40 T antigen.
- To determine how T antigen interacts with DNA replication forks.
- To elucidate the structural basis of T antigen's DNA binding.
Main Methods:
- Utilized synthetic DNA replication fork substrates.
- Employed chemically modified DNA to probe binding interactions.
- Assessed T antigen binding and helicase activity in the presence/absence of ATP.
Main Results:
- T antigen formed an unwinding complex with synthetic replication forks.
- Binding was ATP-dependent and primarily hexameric.
- T antigen exhibited asymmetric binding to one DNA strand, consistent with 3' to 5' helicase activity.
- Recognition involved the sugar-phosphate backbone, with specific phosphate modifications inhibiting activity.
- Binding occurred independently of ATP hydrolysis or a 3' single-stranded end.
Conclusions:
- SV40 T antigen directly binds replication forks through a fork-specific structure.
- The DNA helicase recognizes the DNA backbone at the fork junction.
- These findings provide insights into the molecular mechanism of viral DNA replication.
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