Lecture 16

Question 1

Define the term supercoiling

Answer 1

Supercoil:

• caused by turns in DNA helix
• done to package DNA in nucleus

Prokaryotic supercoiling: negative supercoil
Portein core –> Need:
1. DNA gyrase (Topoisomerase 2) - need ATP
2. DNA topoisomerase 1
3 HU protein

Negative coiling: behind protein
- promote strand separation ie DNA replication

Positive coiling: ahead protein

• introduced during DNA replication and transcription
• needs to be corrected
• make DNA strand separation more difficult

Question 2

Differentiate between a supercoiled and a relaxed molecule.

Answer 2

Supercoiled molecule:

• caused by twisting and turning
• can be relieved in DNA with free end
• introduced by protein and enzymes moving through double helix

Question 3

State the reasons why supercoiling of DNA molecules is important.

Answer 3

Allows for:
1. transcription
2. packaging of DNA in nucleus
3, replication of DNA

Question 4

Compare and contrast DNA packaging in prokaryotes and eukaryotes.

Answer 4

DNA in euk somatic cells:
packaged via histones

Prokaryote:
?

Question 5

Describe the action of DNA gyrase.

Answer 5

DNA gyrase: aka topoisomerase 2

• breaks two backbones
• need ATP

Question 6

List the antibiotic drugs that interfere with DNA gyrase.

Answer 6

Ciproflaxin

Question 7

Describe the structure of chromatin and histone proteins.

Answer 7

DNA –> chromosomes –> chromatin –> DNA + chromosomal protein –> nucleosomes –> nucleosome core –> include linker DNA + H1

• most imp chromosomal protein = histone
• histones = 2 (H2A, H2B, H3, H4)
  - rich in Arg/ R and Lys /K = basic

Question 8

Explain how dsDNA and histones associate to form the nucleosome.

Answer 8

DNA wraps around core particle + linker

Question 9

Describe the different structural conformations of DNA packaging in eukaryotes (10 nm fiber, 30 nm fiber and metaphase chromosomes).

Answer 9

First level of packing:
10 nm: chromatin ‘beads on a string’: nucleosome + linker

Second level of packing:
30 nm: nucleosomes interact with each other between adjacent histone H1 proteins

Most condensed form:
Metaphase = least transcriptionally active

Question 10

Describe how histone modification can influence transcriptional activity.

Answer 10

Heterochromatin: not active; condensed

Euchromatin: active; decondensed

Histone Modification:

1. Acetylation
   - removed positive charge –> reduces affinity between histone + DNA -> increase transcribeption

2) HATs: histone acetyltranserases
- euchromatin
- less condense

3) HDs, or HDACs: histone deacetylases
- nucleosome is more tightly associated with DNA reducing access to DNA

Histone modification: modifications on amino acids found in exposed tail of histones

1. methylation
2. phosphorylation
3. ubitquination

Question 11

Clinical examples utilized in lectures:

i.Inhibitors of DNA gyrase – Quinolone drugs (Ciprofloxacin)

Answer 11

Decitabine: causes DNA hypomethylation and increased transcription