DNA Structure and Topology

Study Questions DNA

1. Understand the difference between the major and minor groove. Specific DNA-binding proteins mainly contact DNA through hydrogen bonds in the major groove of B-DNA. a) Why might sequence-specific binding be more common in the major groove than in the minor groove? b) What hydrogen-bond contacts can proteins make with the bases in the major groove? Are these different from those in the minor groove? 2. Summarize the differences between A-, B-, and Z-DNA. Under what solvent conditions would you expect to find each one? What general sequence of bases is found in segments of DNA that are thought to be in the Z-DNA structure?

Nucleic Acids
1. Be prepared to explain the various forces involved in stabilizing DNA. 2. What are the mechanisms by which flexibility of DNA is constrained? 3. Be able to recognize the various parameters that contribute to DNA's conformational flexibility if you are given structures. (ex: -angle, backbone torsion angles, syn- and anti- steric orientations, nucleotide sugar conformations, etc)

Topoisomerases (Will Not Be Covered During Spring 2007 Lectures)

1. Thoroughly understand the mechanisms of action of Type I (both IA and IB) and Type II Topoisomerases.

2. Distinguish between the "controlled rotation" mechanism and the "strand passage" mechanism and know which mechanism is used for Type IA, Type IB and Type II Topoisomerases. 3. Briefly explain the mechanisms of action of Ciprofloxacin, Novobiocin, Etoposide, and Doxorubicin in blocking topoisomerase activity. 4. Explain the following terms: twist, writhe, super-coiling, linking number and be able to determine these for simple models of DNA. 5. DNA negative supercoils can be titrated out by "unwinding agents." Ethidium bromide, a planar molecule, "intercalates" between the stacked DNA bases, thereby unwinding the supercoils. However, the linking number of the DNA is not changed. Explain the physical basis for the ability of ethidium bromide to "unwind" these supercoils. 6. Eukaryotic DNA does not have a DNA gyrase activity, as does bacteria. How are negative supercoils introduced into eukaryotic DNA such that the DNA can be compacted? 7. DNA Gyrase and DNA Topoisomerase IA have diametric functions in E. coli. Explain. 8. You have discovered an enzyme secreted by a particularly virulent bacterium that cleaves the C2'-C3' bond in the deoxyribose residues of duplex DNA. What is the effect of this enzyme on supercoiled DNA? Explain your answer. 9. A closed circular duplex DNA has a 100-bp segment of alternating C and G residues. On transfer to a solution containing a high salt concentration, this segment undergoes a transition from the B conformation to the Z conformation. What is the accompanying change in its linking number, writhing number and twist? 10. Explain how negative supercoils can be introduced into eukaryotic DNA, despite the fact that eukarytic DNA do not have DNA Gyrase activity. I may provide a diagram of the formation of one wrap of a circular DNA around a histone protein, followed by removal of the compensatory (+) supercoil. You will explain the steps involved, mentioning the enzyme type involved. 11. When the helix axis of a closed circular duplex DNA of 2340 bp is constrained to lie in the plane, the DNA has a twist of 212. When released, the DNA takes up its normal twist of 10.4 bp/turn. Indicate the values of "Lk",

"Wr", and "T" for both the constrained and unconstrained conformational states of this DNA circle.