Module 3 Section 7

Question 1

Chromosome packaging

Answer 1

-End to end length of DNA in diploid human cell ~2m
-nucleus diameter ~5µm
~10 000x reduction in length

Question 2

Chromosome features when stained with Giemsa

Answer 2

Heterochromatin
-dark bands, condensed DNA
-Not transcriptionally active
Euchromatin
-Light regions, less compact DNA
-transcriptionally active regions
Increased darkness=increased condensation=decreased transcriptional activity

Question 3

Telomeres

Answer 3

Sequence (TTAGGG)n
n=800-2500
-hTERT: human telomerase reverse transcriptase, is increased in adult + embryonic stem cells and cancer

Question 4

Functional requirements of DNA compaction

Answer 4

• highly organized
• allow access for factors that regulate replication
• Allow access for factors that regulate transcription

Question 5

What is chromatin

Answer 5

-DNA + protein making up chromosomes

Question 6

Nucleosomes

Answer 6

• unit of chromatin
• DNA wrapped around histone core
• 1.67 turns of DNA around histone, 147 BP
• H1 Subunit: linker histone
• with H1 Linker: 168 BP

Question 7

Histone properties

Answer 7

• assemble into octameric complexes
• pos charge side chain neutralize neg charged DNA backbone (acts like a salt)
• high % of Lys and Arg residues in histones (20-30%), highly conserved over eukaryotes

Question 8

Histone Octamer

Answer 8

• “core” histone subunits
• H2A, H2B form a dimer. Are found 2x in each octamer
• H3, H4 form a tetramer
• requires the presence of DNA and chaperone proteins to assemble

Question 9

Histone Fold motif

Answer 9

• Straight 10 bp segment joined by bends

- motif is 3 a-helices, linked by 2 short loops

Question 10

DNA sequence and nucleosome binding

Answer 10

• 1 turn of double helix = 10BP
• DNA compaction A-T>G-C (b/c weaker H-bonds)
• regions that interact best with histone octamers have A-T pairs 10 BP apart, offset by G-C pairs positioned in between
• A-T pairings in the region where the minor groove of the pairing is facing the histone allows for it to be compressed, bc DNA is naturally bent at A-T pairs

Question 11

N-Terminal histone tails

Answer 11

• Flexible tails
• exit core via alignment of minor grooves every 20 BP
• mediate higher order chromatin structure
• pos charge tails allow for better compaction
• Tail modifications impact nucleosome interactions, changing the degree of chromatin compaction
• Increased interactions=increased DNA access=Increased transcription

Question 12

H1 “linker” histone

Answer 12

-one linker per octamer
-protects additional 21 BP
binding sites:
-DNA linker region (free end)
-central region of nucleosome DNA
-fixes DNA entry/exit angles (no linker= free DNA ends)
-provides extra 6-7x compaction of nucleosomes
-compaction by nucleosomes=6-7x, compaction by H1= 6-7x, total ~36-42x

Question 13

30nm filament

Answer 13

• after binding of H1, nucleosomes condense into filament with 30nm width
• can form without H1
• can’t form without N-term tails of core histones
• seen in vitro, not known if seen in vivo

Question 14

Requirements of DNA compaction system

Answer 14

1. Dynamic
   - rapid changes in condensation so replication/transcription can occur when needed
2. Modifiable
   - globally (ie. replication)
   - locally (ie. transcription of a specific gene)
3. Responsive
   - respond to modification enzymes that alter state of DNA condensation
   - specific regions need to be recognizable by the enzymes

Question 15

What classes of enzymes allow regulation of DNA compaction to be achieved

Answer 15

1. Chromatin remodelling complexes
   - these change location and variant content of octamers
2. Histone modifying enzymes
   - they modify the N-term regions (ie. acetylate lysine)