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 turnswrithe (Wr) = # of times the helix turns on itselflinking 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, 5th 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:

– α2β2 heterotetramer (GyrA2GyrB2)– binds 140 bp DNA– GyrA-CTD wraps DNA– GyrB-NTD ATPase, N-gate (entry)– GyrA-NTD C-gate (exit)

Figure 1 DNA 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 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

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)

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 pNwrapping 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 4 Gyrase activity at high forcesDoes 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 moderelaxation 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 6 Experiments 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 6 Gyrase 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 7 Branched model for gyrase activity

dominates at low force

dominates at high force

dominates at high negative torque

Figure 7 Force-Velocity curves and proposed mechano-chemical model

where:n= α, χ, or p

kn= rate at zero F and τΔxn= extension distance to transition state Δθn= twist angle to transition state

RL= rate limiting step

rising phase due to dependence of kα, RL on torque

zero-order kχ phase

decrease first by kα sensitivity to forcethen by competition with kp (-) 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