Klemm, R. D., Austin, R. J., and Bell, S. P. (1997) Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell 88: 493-502.
1. How were the ATP binding experiments performed? What features of the ATP binding properties of ORC proteins do they rely on?
2. Why did ARS DNA increase ATP binding to ORC by only 2-fold (Fig. 1A)? How does the observation that 0.6 molecules of ATP were bound per ORC in the presence of DNA complicate the interpretation?
3. Fig. 2 is captioned: ORC has multiple ATP-binding sites. What exactly did the authors observe, as shown in panel C, and did the figure support multiple, as opposed to two, binding sites?
4. Why was it inferred that Orc4p does not bind ATP even though azido-modified ATP crosslinked to this subunit?
5. Why do high concentrations of ATP enhance DNA binding by the ORC 1A mutant? How might the nature of the mutation affect this result?
6. Fig. 5 at face value could have been interpreted as meaning that hydrolysis/ binding of ATP by Orc1p then stimulates binding of ATP by ORC 5p. How was this possibility effectively ruled out?
7. What evidence suggests that ATP binds more tightly to Orc1p than to Orc5p?
8. How would you test the allosteric cross-subunit mechanism for ATP-mediated DNA binding proposed by the authors?
9. What experiments can you think of to distinguish between models I, II and III as presented in Fig. 7?
10. Can you think of other cases in which small ligands affect DNA-binding affinity? How do the mechanisms compare to those proposed here?