Lecture 19 - DNA and RNA Structure 2

Question 1

How can you check denaturation and reannealing

Answer 1

Through absorbance spectra of A260. stacked bases absorb less UV light than unstacked. look at hyperchromatic shift. A-T is a weaker bond, so melts first, can be prevented by adding salt concentration (prevents phosphates from repelling) and longer sequences to stabilize duplex.

Question 2

What happens when DNA is strained?

Answer 2

Either undergoes supercoiling or else breaks H-bonds, less favourable b/c of amount of energy needed to break bonds
Actively underwind DNA to induce supercoiling and store energy

Question 3

Define Lk, how can it be changed

Answer 3

Linking number = bases/bp/turn (# turns)
Lk>0 = right handed helix
Lk < 0 = left handed helix
Can only be changed by breaking and rejoining one or both strands
ΔLk < 0 = underwound supercoil = negative = right handed
ΔLk > 0 = overwound supercoil = positive = left handed
Supercoiled DNA is much more compact, moves through gel electrophoresis quicker

Question 4

What molecule can change Lk?

Answer 4

Topoisomerases
Topo 1 –> increases Lk by 1, can relax negative supercoils, nicks one strand of DNA and passes other through ss break, thermodynamically favourable
Topo 2 –> changes Lk by 2, can relax or introduce negative supercoils, breaks both strands of DNA, uses energy

Question 5

How do topoisomerases generally work?

Answer 5

1) Cleave 1 or 2 strands of ds DNA
2) pass segment of DNA through break
3) reseal DNA breaks