Since amplification of the wt strand is linear (one new strand made per cycle), there are 2+3wt strands afier 3 cycles and 2+7wt strands afier 7 cycles. The total number of strands is 24=16 afier 3 cycles and 28=256 after 7 cycles. 3/16=18.75%and 9/256=3.5% 4. Explain the difference between plectonemic and solenoidal supercoiling 5. You have isolated a circular plasmid from E Coli. and analyze it on an agarose gel which you stain with ethidium bromide after electrophoresis a)What is the topological state of the plasmid and why? Where does it migrate on the gel (lane It is negatively supercoiled due to bacterial gyrase in the cell so migrates at the position of sc DNA b)Now you run the gel in presence of the drug netropsin which binds in the minor groove of DNA and locally increases the twist of the double helix (Snounou and Malcom, 1983, JMB 167 211). Ind icate where it migrates relative to a plasmid separated in the absence of the drug(lane Since the drug locally overnwists the DNA, it will cause unwinding elsewhere and the plasmid will adopt negative writhe. Since it is already negatively supercoiled, it willyjust become more so, and its position in the gel won't change c) Now you incubate the plasmid in the presence of netropsin and topoisomerase I. You then use phenol/chloroform to separate the plasmid from the drug and the topo, and you run it on a gel with buffers that lack the drug. How do you pred ict it would run in the presence of low amounts of drug(during the incubation with topo D(lane 3)and high amounts of drug(lane 4)? Netropsin locally overnwists the DNA, causing compensatory negative supercoiling. Topo I relaxes the negative supercoils but doesnt displace the drug, so after extraction you have dNa that is overtwisted relative to the starting material(another way to say it is that you have reduced the linking number). I you add small amounts of the drug, you will remove a few of the negative supercoils originally present in the plasmid, so the treated plasmid will run less rapidly than the original (lane 3), but if you add enough of the drug, you will add enough positive supercoils that the plasmid once again migrates at the position of supercoiled DNA (lane 4) d)In which case a-c did your experimental manipulation change the linking number of the plasmid relative to when it was isolated from the cells? What about writhe? Linking number only changed in(c), but the writhe changed in(b) and(c) 6. Is a purine-pyrimid ine or a pyrimid ine-purine stack more likely to undergo roll? Explain Pyr-Pur more likely to roll because the base-stacking interactions are fewer. 7. You have just determined the crystal structure of Y FDB (your favorite DNA binding protein), and the interactions it makes with the phosphate backbone of dNa pred icts it will displace 4 counter ions upon bind a)Calculate the free energy of bind ing that comes from ion displacement in 75 mM KCI and express your result as a dissociation constant some useful equationsSince amplification of the wt strand is linear (one new strand made per cycle), there are 2+3 wt strands after 3 cycles and 2+7 wt strands after 7 cycles. The total number of strands is 24 = 16 after 3 cycles and 28 = 256 after 7 cycles. 3/16 = 18.75% and 9/256 = 3.5%. 4. Explain the difference between plectonemic and solenoidal supercoiling. 5. You have isolated a circular plasmid from E.Coli. and analyze it on an agarose gel which you stain with ethidium bromide after electrophoresis. a) What is the topological state of the plasmid and why? Where does it migrate on the gel (lane 1)? It is negatively supercoiled due to bacterial gyrase in the cell so migrates at the position of SC DNA b) Now you run the gel in presence of the drug netropsin which binds in the minor groove of DNA and locally increases the twist of the double helix (Snounou and Malcom, 1983, JMB 167, 211). Indicate where it migrates relative to a plasmid separated in the absence of the drug (lane 2). Since the drug locally overtwists the DNA, it will cause unwinding elsewhere and the plasmid will adopt negative writhe. Since it is already negatively supercoiled, it will just become more so, and its position in the gel won’t change. c) Now you incubate the plasmid in the presence of netropsin and topoisomerase I. You then use phenol/chloroform to separate the plasmid from the drug and the topo, and you run it on a gel with buffers that lack the drug. How do you predict it would run in the presence of low amounts of drug (during the incubation with topo I) (lane 3) and high amounts of drug (lane 4)? Netropsin locally overtwists the DNA, causing compensatory negative supercoiling. Topo I relaxes the negative supercoils but doesn’t displace the drug, so after extraction you have DNA that is overtwisted relative to the starting material (another way to say it is that you have reduced the linking number). If you add small amounts of the drug, you will remove a few of the negative supercoils originally present in the plasmid, so the treated plasmid will run less rapidly than the original (lane 3), but if you add enough of the drug, you will add enough positive supercoils that the plasmid once again migrates at the position of supercoiled DNA (lane 4). d) In which case a-c did your experimental manipulation change the linking number of the plasmid relative to when it was isolated from the cells? What about writhe? Linking number only changed in (c), but the writhe changed in (b) and (c). 6. Is a purine-pyrimidine or a pyrimidine-purine stack more likely to undergo roll? Explain. Pyr-Pur more likely to roll because the base-stacking interactions are fewer. 7. You have just determined the crystal structure of YFDB (your favorite DNA binding protein), and the interactions it makes with the phosphate backbone of DNA predicts it will displace 4 counter ions upon binding. a) Calculate the free energy of binding that comes from ion displacement in 75 mM KCl and express your result as a dissociation constant. some useful equations: