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Local unwinding during replication results in overwinding or supercoiling of surrounding regions

DNA topology. Local unwinding during replication results in overwinding or supercoiling of surrounding regions. From the field of topology: twist (Tw) = # of dsDNA turns writhe (Wr) = # of times the helix turns on itself linking number (Lk) = sum of twist and writhe. Lk = Tw + Wr.

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Local unwinding during replication results in overwinding or supercoiling of surrounding regions

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  1. DNA topology Local unwinding during replication results in overwinding or supercoiling of surrounding regions From the field of topology: twist (Tw) = # of dsDNA turns writhe (Wr) = # of times the helix turns on itself linking number (Lk) = sum of twist and writhe Lk = Tw + Wr Molecules that differ only by Lk are topoisomers of eachother. Lk can only be changed by breaking covalent bonds Adding 1 negative supercoil reduces Lk by 1

  2. DNA topology Wasserman & Cozzarelli, Science 1986 Two types of supercoiling Biochemistry, 5th ed. Berg, Tymoczko, Stryer

  3. Topoisomerases Reduce supercoiling strain by changing the linking number of supercoiled DNA Type I topoisomerases: - produce transient single-strand breaks (nicks) - remove one supercoil per cycle - changes linking number by 1 or n - ATP-independent - examples= topo I, topo III, reverse gyrase Type II topoisomerases: - produce transient double-strand breaks - remove both positive and negative supercoiling - changes linking number by +/- 2 - ATP-dependent - examples= topo II, topo IV, DNA gyrase

  4. Strand passage by topoisomerases e.g. DNA Gyrase Corbett KD & Berger JM (2004) Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct 33, 95–118.

  5. DNA Gyrase • one of two E. coli type II topoisomerases • relaxes (+) supercoils • introduces (–) supercoils • exhibits ATP-independent (–) supercoil relaxation • Structure: • α2β2 heterotetramer (GyrA2GyrB2) • binds 140 bp DNA • GyrA-CTD wraps DNA • GyrB-NTD ATPase, N-gate (entry) • GyrA-NTD C-gate (exit)

  6. Figure 1 DNA Gyrase mechanism of action model for introduction of (-) supercoils: “α mode” G and T proximal This model does not account for other activities of gyrase - (+) and (-) supercoil relaxation - decatenation - passive relaxation and the dependence on force and torque in the experiments

  7. Figure 2 Magnetic tweezers experimental setup • 15.7 kb DNA molecule with biotinylated or digoxigenated ends • 4 mM MgCl2, 1 mM ATP • supercoiling quantitatively introduced by rotation of magnets • change in bead position monitored by comparing calibrated diffraction ring patterns

  8. Figure 3 Gyrase activity at low forces Starting with (+) supercoiled DNA obs: DNA extended (supercoiling relaxed) Starting with (+) supercoiled DNA at slightly lower force, obs: DNA extended (supercoiling relaxed), then (-) supercoiling introduced (DNA shortened)

  9. Figure 4 T-segment G-segment Gyrase activity at high forces Starting with (+) supercoiled DNA at high tensions: obs: processive relaxation can occur at high force (tension). velocity independent of force between 1.5 – 4.5 pN wrapping independent mechanism 4.5 pN 2.5 pN “χ- mode” activity “distal T-capture” where G-segment and T-segment are not proximal i.e.: discontinuous DNA segments juxtaposed by plectonemic crossings

  10. Figure 4 Gyrase activity at high forces Does high force (+) relaxation require (+) crossings? (test of “χ-mode” model) Experiment: 110 (+) supercoils introduced, then allowed to be relaxed by gyrase. Then, 110 new supercoils introduced while monitoring length. Observation: Linear decrease in extension, indicates DNA not relaxed past buckling transition Consistent with χ-mode relaxation buckling transition High force relaxation requires plectonemic crossings (distal T-segments)

  11. Passive relaxation mode supp fig 3 Figure 5 relaxation in the absence of ATP Start with (-) supercoiled DNA, gyrase, no ATP obs: processive relaxation at moderate forces. Requires high concentrations of gyrase (20 nM vs 1 nM) Relaxation observed only for (-) supercoils, and requires plectonemic DNA. (+) supercoil relaxation experiment not shown Modulation between modes by force blue= high force passive relaxation of (-) supercoils yellow = low force α-mode ATP-dependent introduction of (-) supercoils p-mode requires plectonemes ATP does not stimulate (-) supercoil relaxation at forces that inhibit α-mode (0.6 pN)

  12. Important observation: not stimulated by ATP Three distinct modes observed • α-mode: (+) supercoil relaxation, (-) supercoil introduction • ATP-dependent • wrapping mediated • inhibited by high force • proximal T-segment capture • χ-mode: (+) supercoil relaxation • ATP-dependent • wrapping independent • processive at high force • distal T-segment capture • requires (+) plectonemes • Passive mode: (-) supercoil relaxation • ATP-independent • requires (-) plectonemes • processive at forces that inhibit α-mode

  13. Figure 6 Experiments with DNA braids DNA braids allow more direct measurements of plectonemic associated modes • Functional predictions: • Under high force to inhibit wrapping, χ-mode activity should unbraid L-braided DNA (identical to (+) supercoils) • (-) supercoil relaxation strictly ATP-independent suggests chiral preference for distal T-segment capture, thus R-braids should not be relaxed

  14. Figure 6 Gyrase unbraiding DNA 1 mM L-braids (+) supercoils R-braids are not a substrate for gyrase regardles of ATP, enzyme or force. Gyrase rapidly and completely unbraids L-braids ATP-dependently Braids have zero torque. Indicating that passive-mode relaxation requires negative torque

  15. Putting it all together: Mechanochemical modeling

  16. Figure 7 Branched model for gyrase activity dominates at low force dominates at high force dominates at high negative torque

  17. Figure 7 Force-Velocity curves and proposed mechano-chemical model where: n= α, χ, or p kn= rate at zero F and τ Δxn= extension distance to transition state Δθn= twist angle to transition state rising phase due to dependence of kα, RL on torque zero-order kχ phase decrease first by kα sensitivity to force then by competition with kp (-) sc introduction RL= rate limiting step

  18. DNA Gyrase operates in three distinct modes • Explains prior puzzling observations • gyrase “slippage” uncoupling of ATP hydrolysis from (-) sc relaxation • Distal T-capture explains how gyrase can relax circles smaller than the minimum wrapping size • explains the low-level decatenation in vivo • decatenase activity stimulated by tension forces • conditional lethality of segregation defects rescued by SetB overexpression  SetB induces DNA tension

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