DenaturationRenaturation

1
Denaturation/Renaturation

• Denaturation separation of the 2 strands of the
  duplex (H bonds are broken)
• Denaturants high temperature
• high pH
• low salt
• non-polar solvents (e.g. DMSO)
• Renaturation rejoining of 2 strands
• also called annealing

2
Denaturation/Renaturation
3
Hyperchromic Shift(evidence of denaturation)
Hyperchromic shift
4
GC Content and Salt Affect Tm
Low salt
High salt
5
Linear vs Circular DNA

• Linear DNA eukaryotic nuclear DNA
• However, linear DNA is fixed into loops by DNA
  binding proteins within the chromosome
• Circular DNA most viral and bacterial chromosomes

6
cccDNA

• Covalently closed circular DNA
• Topologically constrained

Relaxed Supercoiled
7
Topological Properties of cccDNA

• Twist (Tw) of times 2 strands cross each
  other
• Writhe (Wr) of times helix crosses over
  itself in supercoiled DNA
• Linking Number (Lk) Tw Wr

- Wr ? negatively supercoiled Wr ?
positively supercoiled
8
Writhe
Interwound or plectonemic
Toroid or spiral
Example DNA wraps around histone protein core
within nucleosomes Left-handed spiral
9
Linking Difference

• Lk0 linking number for relaxed B DNA at
  physiological conditions
• Linking difference a measure of supercoiling
• DLk Lk Lk0
• Wr!
• - DLk ? negative supercoiling (underwound)
• DLk ? positive supercoiling (overwound)

10
Superhelical Density

• Number of supercoils/helical turn
• s DLk/Lk0
• Wr/Tw

11
Physiological Supercoiling

• DNA in proks and euks tends to be negatively
  supercoiled (s -0.06)
• Advantage Strands of underwound DNA are easier
  to separate (facilitates replication,
  transcription)
• DNA in thermophiles is positively supercoiled
• Advantage Strands of overwound DNA are harder
  to separate (avoids threat of denaturation in
  high temps)

12
Topoisomerases

• Type I produce single strand breaks
• Type II produce double strand breaks
• Relieve supercoiling (both positive and negative)

13
Type I Topoisomerases

• Removal of a negative supercoil
• Increases the Lk by 1
• What happens in cases where type I topoisomerase
  removes a positive supercoil?

14
Type II Topoisomerases

• Can also relieve positive or negative supercoils
• Change the linking number in increments of 1

15
Topoisomerases as Detanglers
16
Mechanism of Type I Topoisomerase Action
17
Mechanism of Type I Topoisomerase Action
18
Supercoiled DNA Migrates Faster on DNA Gels