Local unwinding during replication results in overwinding or supercoiling of surrounding regions DNA topology Lk = Tw + Wr 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 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

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

Topoisomerases 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 Reduce supercoiling strain by changing the linking number of supercoiled DNA

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

DNA Gyrase one of two E. coli type II topoisomerases relaxes (+) supercoils introduces (–) supercoils exhibits ATP-independent (–) supercoil relaxation Structure: –α 2 β 2 heterotetramer (GyrA 2 GyrB 2 ) –binds 140 bp DNA –GyrA-CTD wraps DNA –GyrB-NTD ATPase, N-gate (entry) –GyrA-NTD C-gate (exit)

Figure 1DNA Gyrase mechanism of action model for introduction of (-) supercoils: “α mode” 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 G and T proximal

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

Figure 3Gyrase 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)

Figure 4 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 “ χ - mode” activity “distal T-capture” where G-segment and T-segment are not proximal i.e.: discontinuous DNA segments juxtaposed by plectonemic crossings G-segment T-segment 2.5 pN 4.5 pN

Figure 4Gyrase 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)

Figure 5 Passive relaxation mode relaxation in the absence of ATP 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 supp fig 3 ATP does not stimulate (-) supercoil relaxation at forces that inhibit α-mode (0.6 pN) Start with (-) supercoiled DNA, gyrase, no ATP obs: processive relaxation at moderate forces. p-mode requires plectonemes

Three distinct modes observed 1.α-mode: (+) supercoil relaxation, (-) supercoil introduction -ATP-dependent -wrapping mediated -inhibited by high force -proximal T-segment capture 2.χ -mode: (+) supercoil relaxation -ATP-dependent -wrapping independent -processive at high force -distal T-segment capture -requires (+) plectonemes 3.Passive mode: (-) supercoil relaxation -ATP-independent -requires (-) plectonemes -processive at forces that inhibit α-mode Important observation: not stimulated by ATP

Figure 6Experiments with DNA braids DNA braids allow more direct measurements of plectonemic associated modes Functional predictions: 1.Under high force to inhibit wrapping, χ -mode activity should unbraid L-braided DNA (identical to (+) supercoils) 2.(-) supercoil relaxation strictly ATP-independent suggests chiral preference for distal T-segment capture, thus R- braids should not be relaxed

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

Putting it all together: Mechanochemical modeling

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

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

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