You are given the following facts:
(i) The sequence of the binding site of a protein is:
T1 G2 T3 G4 A5 G6 A7 T8 A9 T10 C11 T12 C13 A14 C15 A16
A1 C2 A3 C4 T5 C6 T7 A8 T9 A10 G11 A12 G13 T14 G15 T16
(ii) The bold region of the DNA is B-form, both free in solution and in complex with the protein.
(iii) DMS is a chemical probe experiments that adds a methyl group to exposed N7 of guanines and the N3 of adenines. The italicized residues are protected from DMS methylation in the presence of the protein.
(iv) A mutation that changes base pairs 8 and 9 from A:T-T:A to G:C-C:G decreases the affinity of the protein for the DNA.
(v) Inosine, structure of which is identical to guanine, except it lacks the N2 amino group, forms Watson-Crick base pairs with cytosine. Substituting I:C base pairs for the A:T base pairs at position 8 and 9, does not affect the affinity of the protein for its binding site.
(vi) In the presence of the protein, base pairs 8 & 9 become susceptible to modification by a reagent (diethylpyrocarbonate-DEPC) that only modifies unpaired bases.
4pts a. Which surface of the DNA helix contains the atoms that are protected from DMS methylation by the protein.
2pts b. What does the increased sensitivity of the DNA to DEPC modification in the presence of the protein indicate? (1-2 sentences).
5pts c. Considering all facts (and the structure of adenine, inosine and guanine) explain why G:C base pair substitution decreases binding, while inosine has no effect on binding (4 sentences).
7pts d. Predict the result of the following experiment: A linear piece of DNA containing the protein binding site was ligated to form a circle in the absence and presence of protein. After removing the protein, the two circular DNA samples were electrophoresed on an agarose gel. Would you expect the two samples to have the same or different topoisomer distributions? Why? Explain your answer. Describe an experiment that you can do to test your explanation.