5. DNA supercoiling and proteins

if both ends are fixed = situation in circular dna molecules and locally in long linear dna molecules (bound by chromatin scaffold)

strain is released by writhing into superhelical turns (supercoils)

one dna supercoil forms in the double helix for every

10 bp opened for b dna

positive supercoil

two strands in FRONT of the opening become wrapped around each other more than once every 10 bp - overwinding (turns are shorter - fewer bases/new turn)

consequence of positive coil

occurs when right-handed B-dna is twisted really tightly about its axis, the double helix begins to distort and knot (stress) into positive left-handed supercoils

negative supercoils

two dna strands behind the opening become wrapped around each other less than once every 10 bp - underwinding (turns are “longer” more bases/new turn)

consequence of negative supercoils

“loosing” the tension and causing the B-dna helix to start un-twisting/unwinding there by increasing stress leading to negative right-handed supercoils

Twisting # - T

the crossing of one strand of dsDNA over the other, it measures how tightly the helix is wound

for a 2,000 bp dna duplex, (we assume its a normal b dna)

t = 200 bc 2000 bp/ 10bp/turn = 200 turns

if right hand helix

T is positive

if left hand helix (z)

t is negative

Writhing # - W

nnumber of superhelical turns; refers to the twisting of the dsdna axis in space (how many times the duplex DNA crosses over itself)

Relaxed dsDNA

w=0

left supercoils

W is negative

right supercoils

W is positive

Linking %

total # of times one strand of closed molecule of dsDNA encircles the other strand (integer). reflects both the twisting of the native dna helix and the presence of any supercoiling (w)

L =

T + W

L can only be changed by breaking

one or both strands of the DNA, winding them tighter or looser, and rejoining the ends = change W

L is a constant in unbroken duplex DNA

so any change in T must be accompanied by an equal and opposite change in supercoiling

most cell dnas are ______

negatively supercoiled by they store energy, energy can be converted into untwisting of double helix

dna overwound

positive supercoiling - reduced chance for dna-protein interaction

dna underwound

negative supercoils store energy that could help strand separation - untwisting favoured ( important for replication and transription)

prokaryotic Topoisomerase I

nicking-closing enzyme, makes transient cuts in one strand, relaxes negative supercoiling in prokaryotes

prokaryotic Topo II

relazes positive supercoiling (uses ATP) makes double-stranded cut, pass a duplex DNA thorugh it and reseals the cut. changes L # in steps of 2

prokaryotic gyrase one of bacterial topo II

introduces negative supercoils

prokaryotic reverse gyrase. topo 1 generating positive supercoils (requires ATP)

1. stabilizing the genome structure at high temp

2. protecting the dna strand breakage promoted by exposing dna to high temperature

eukaryotic top1, top1mt

topo I action, relaxes both positive and negative supercoils. single strand clevage. top1 is found in the nucleus, top1mt in mitochondria

eukaryotic top2alpha, top2beta

topo II action only relaxes sueprcoiled DNAs - cannot induce supercoils, works on both positive and negative supercoils (decatenate) , nuclear and mitochondrial

eukaryotic top3 alpha, top3 beta

topoisomerase I activity, only relaxes hypernegative supercoiling (HSc-) requires magnesium. top3beta cna also act as an rna helicase