Supercoiling of DNA 1 Topology A Right handed

Supercoiling of DNA 1. Topology A. Right handed supercoiling = negative supercoiling (underwinding) B. Left handed supercoiling = positive supercoiling C. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteins E. Unraveling the DNA at one position changes the superhelicity F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA 1. Topology A. Right handed supercoiling = negative supercoiling (underwinding) B. Left handed supercoiling = positive supercoiling C. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteins E. Unraveling the DNA at one position changes the superhelicity F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA 2. Numerical expression for degree of supercoiling A. Equation Lk=Tw+Wr B. L: linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist, # of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoiling E. specific linking difference or superhelical density=DLk/Lk 0

Supercoiling of DNA 2. Numerical expression for degree of supercoiling A. Equation Lk=Tw+Wr B. L: linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist, # of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoiling E. specific linking difference or superhelical density=DLk/Lk 0

Supercoiling of DNA 2. Numerical expression for degree of supercoiling A. Equation Lk=Tw+Wr B. L: linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist, # of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoiling E. specific linking difference or superhelical density=DLk/Lk 0

Supercoiling of DNA 1. Topology A. Right handed supercoiling = negative supercoiling (underwinding) B. Left handed supercoiling = positive supercoiling C. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteins E. Unraveling the DNA at one position changes the superhelicity F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA 3. DNA compaction requires special form of supercoiling A. Interwound: supercoiling of DNA in solution B. Toroidal- tight left handed turns, packing of DNA both forms are interconvertible

Supercoiling of DNA 4. Methods for measuring supercoiling based on how compact the DNA is A. Gel electrophoresis i. 1 dimensional ii. 2 dimensional B. Density sedimentation

Supercoiling of DNA 4. Topoisomerases are required to relieve torsional strain A. Topoisomerases I : breaks only one strand B. Topoisomerase II : breaks both strands

Supercoiling of DNA 4. Topoisomerases are required to relieve torsional strain A. Topoisomerases I - breaks only one strand i. monomeric protein ii. after nicking DNA the 5'-PO 4 is covalently linked to enzyme (prokaryotes) or the 3' end is linked to the enzyme (eukaryotes) iii. evidence is the formation of catenates iv. E. coli Topo I relaxes negatively supercoiled DNA v. introduces a change of increments of 1 in writhe

Supercoiling of DNA 4. Topoisomerases are required to relieve torsional strain B. Topoisomerase II - breaks both strands i. supercoils DNA at the expense of ATP hydrolysis ii. two subunits: (alpha)2 and (beta)2 iii. becomes covalently linked to the alpha subunit iv. relaxes both negative and positively supercoiled DNA v. introduces a change in increments of 2 in writhe.