| Abstract: |
The ATP-dependent binding of the simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication (ori) results in the structural distortion of two critical elements within flanking regions of ori and the untwisting of the DNA helix. We examined the effect of changes to temperature, ATP concentration, and other reaction parameters on the generation of these DNA structural changes. We found that induction of the two localized structural transitions were highly and differentially sensitive to reaction conditions. Significant distortion of the early palindrome element, shown previously to result from DNA melting, required low levels of ATP (10 to 30 μM) but temperatures above 25°C. Distortion of the AT tract occurred at low temperatures (5°C) but required relatively high concentrations of ATP (>300 μM). Thus, T antigen can induce structural changes within one critical element of ori without generating significant structural distortion within the second element. The response of ori untwisting to reaction conditions generally increased in parallel with or fell intermediate between the inductions of localized structural transitions. We suggest that ori untwisting and localized structural distortions are interdependent consequences of T-antigen binding to ori. These results suggest a model for the structural events occurring during the initial steps of SV40 DNA replication. |
