MBB222 - Lecture 21: DNA/RNA structure

What is DNA supercoiling

overwinding or underwinding of a DNA strand

What does supercoiling do to DNA

creates torsional strain → forces the molecule to coil back on itself

Why is DNA supercoiling essential

• important for compacting genetic material inside cells

• regulates the accessibility of DNA during DNA replication/transcription

what does topoisomerase do

• relieves twisting tension

• removes knots and tangles

• prevents over coiling during processes like replication and transcription

when can supercoiling exist

can only exist in a DNA molecule where both strands of DNA are closed circles/fixed at one end → topologically constrained double stranded

why does DNA need to be topologically constrained in order for supercoiling to occur

must be topologically constrained so that its linking number (Lk) stays constant

What would happen to Lk if DNA wasn’t topologically constrained

any torsional stress or untangling inflicted on the DNA strand would lead to the energy being released instead of it forcing the double helix to coil upon itself

What would happen if a strand breaks when supercoiling occurs

if a strand breaks - the DNA would rapidly lose it’s supercoiling and reach a relaxed state

what are topoisomers

DNA molecules that have different coiled forms but have the same nucleotide sequence

what is topology

the study of the 3D shape + structural properties of molecules that remain unchanged when bent/twisted without breaking it’s chemical bonds

what is linking number

number of times one strand would have to be passed through the other strand in order for the strands to be completely separated from each other

what is the relation equation for Lk/Tw/Wr

Lk = Tw + Wr

what is twist (Tw)

the geometrical property of the helix → a trait/characteristic that describes the shape/geometric figure

how is twist (Tw) defined for dsDNA

Tw is the number of full turns of the helix

how would you determine twist (Tw) if given the amount of base pairs

total base pairs divide by 10.5 base pairs (B form → 10.5 bp = 1 twist)

what is writhe (Wr)

measures the degree of supercoiling → number of times a double helix crosses itself

if a ds helix writhes in the left direction - what kind of winding/what is the value of Wr (±)

if in left direction:

• overwinding

• writhe has a positive value

if a ds helix writhes in the right direction - what kind of winding/what is the value of Wr (±)

• under winding

• writhe has a negative value

why does under-winding make it easier to separate the strands of a double helix

under-winding stores up torsional strain (negative supercoiling) → acts as stored mechanical energy → lowers the energy barrier needed to melt the hydrogen bonds between the bases separating the strands

why does over-winding make it harder to separate the strands of a double helix

over-winding increases torsional strain → increases the amount of energy needed in order to break the bonds between the bases → requires more energy in order to separate the strands

why is linking number (Lk) a topological property of DNA

Lk is a topological property as the value can’t be changed unless one or both strands of the duplex is broken

compare supercoiled and relaxed DNA

supercoiled:

• alters the overall form of DNA

• cannot lay flat on a planar → has extra twists

• has a higher folding of a secondary structure

• more compact → moves faster in centrifugation / gel electrophoresis

relaxed:

• relaxed DNA can lay flat on a planar surface

• less compact

• moves slower in centrifugation / gel electrophoresis

how is DNA observed in gel electrophoresis

DNA can be stained with nucleic acid staining dyes → ethidium bromide (EthBr) / SYBER green

• fluorescent dyes insert themselves between the base pairs → stains them

• the stained DNA fluoresces under a UV lamp

how does topoisomerase change the linking number of DNA

cleaves one/both strand(s) → passes another DNA strand through the gap → reseals the gap

what are the difference between topoisomerase I / topoisomerase II

topoisomerase I

• breaks 1 strand of DNA → other strand passes through the gap

• adds/removes Wr by increments of 1

• changes Lk by increments of 1

• thermodynamically favoured → driven by the energy released from supercoiling

topoisomerase II

• breaks both strands

• adds/removes Wr by increments of 2

• changes Lk by increments of 2

• can relax negative/positive supercoils

• thermodynamically favourable → still requires ATP energy input

what happens when topoisomerase is not present

cells cannot:

• replicate

• package their DNA

• express their DNA

result: cells die

what is quinolones

an antibiotic that inhibits bacterial DNA gyrase ( a type II topoisomerase ) → blocks the last step (resealing) of the topoisomerase reaction → stops/pauses DNA replication

what is ciprofloxacin

an antibiotic that targets topoisomerase that are present in tumour cells → targets type I / type II topoisomerase → reduces the growth/replication of tumour cells