Dowell, S.J., P. Romanowski, & J.F.X. Diffley (1994) Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science 265: 1243-1246.
1. Describe the ARSl-lacZ reporter gene. A diagram would be helpful.
2. The authors propose to use the ARSl-lacZ reporter to identify proteins that bind to origins of replication.
(a) Describe the concept of this approach which is sometimes referred to as 'the one-hybrid' method.
(b) Does a positive result in this case indicate direct binding of the library fusion protein to ARS1, and if not, why?
(c) Would you think differently if you were using a mouse ORI and a mouse fusion library? Explain.
3. Figure 1C suggests that Dbf4p interacts generally with yeast origins. What experimental result is the basis of this conclusion?
4. The results in Figure 2 indicate that Dbf4p binds preferentially to domain A of the ARS1. Why do the authors suggest that this binding is not directly to the DNA but is mediated by ORC?
5. The reporter gene in this study is lacZ and therefore expression can be monitored on plates using X-gal and blue color production. Based on the results reported in Figure 4, mark the regions containing the ARS1-binding site and the Cdc7p-binding site on the rectangular bar labeled FL in the diagram below. FL represents the full-length Dbf4 protein.
6. Draw a diagram showing the structure of the ARS1, ORC, Cdc7p, Dbf4p complex indicating clearly which components are interacting with which other components based on the results reported in this article.
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