DNA Topology Handout - Cmgm Stanford

DNA TopologyNeither of these methods bythemselves can distinguish negativelyand positively supercoiled DNA. Bothforms sediment and electrophoresemore rapidly than relaxed DNA.However by sedimenting orelectrophoresing supercoiled DNA inthe presence of an intercalating agentsuch as ethidium bromide orchloroquine, one can distinguishnegatively supercoiled DNA frompositively supercoiled DNA. Whennegatively supercoiled DNA binds anintercalating agent, the average pitch isreduced because the twist anglebetween adjacent base pairs on eitherside of the intercalating agent isreduced from 36° to as little as 10° of twist.This reduction of twist causes acompensatory increase in writhe in acovalently closed molecule. Hence amolecule that is initially negativelysupercoiled will become more relaxed and apositively supercoiled molecule willbecome more twisted. By electrophoresingor sedimenting supercoiled DNA inincreasing concentrations of anintercalating agent and knowing theaffinity of the agent for DNA it is possibleto calculate the writhe in the DNA.It is also possible to measure thewrithe and the sense of the write usingintercalating agents in 2 dimensional gelelectrophoresis experiments. By firstelectrophoresing DNA in one dimensionin the absence of intercalating agents asabove, and then adding an intercalatingagent to partially increase the writhe in apositive sense and electrophoresing theDNA in a second dimension, it is possibleto separate positive and negatively writhedDNA in the dimensions. It is also possible to get much better resolution of the highlytwisted forms of DNA found in natural supercoiled DNA samples.Measuring the Pitch of DNASince the superhelical state of DNA is dependent on the pitch of DNA it becamecritical to have an accurate method to determine a DNAs pitch under different-6-