Lecture 16

Question 1

Why is DNA condensed?

Answer 1

It would be too long to fit into cells otherwise

Question 2

What is the hierarchy between chromatin, chromosomes, and nucleosomes?

Answer 2

Nucleosome -> chromatin -> chromosomes

Question 3

What are the different levels of compaction? List the length scale associated with them.

Answer 3

Short region of DNA double helix - 2nm
“beads-on-a-string” form of chromatin - 11nm
30-nm chromatin fiber - 30nm
section of chromosome in extended form - 300nm
condensed section of chromosome - 700nm
entire mitotic chromosome - 1400nm

Question 4

Approximately how many bp of DNA per nucleosome?

Answer 4

1 nucleosome = 200bp DNA

Question 5

What is a nucleosome?

Answer 5

• Nucleosome is the basic unit of DNA packaging formed by wrapping DNA around the histones
• only forms in eukaryotes

Question 6

What is chromatin?

Answer 6

Beads-on-a-string with the beads being individual nucleosomes. This is the conformation that is most often observed

Question 7

What is a chromosome?

Answer 7

The most condensed form and can be observed in meiosis or mitosis

Question 8

What is the experimental evidence of nucleosome repeat length?

Answer 8

Using micrococcal nuclease, because its digestion is not affected by the age of the cell or culturing state DNA bound by histones cannot be cut. From this assay 400, 600,800,… fragments result and so conclude that the nucleosome repeat length in human is ~200bp

Question 9

How many histones are there and what is their charge? What is the cause of this charge on histones?

Answer 9

Five. High abundance of positively-charged amino acids (Lysine and Arginine)

Question 10

Oder the histone molecules from least to greatest conservation

Answer 10

H1 < H2A/H2B < H3/H4

Question 11

Explain nucleosome assembly.

Answer 11

1. The assembly of a nucleosome is initiated by the formation of H3(2)-H4(2) tetramer
2. Tetramer binds to dsDNA
3. Bound tetramer recruits H2A-H2B dimer to complete the assembly of the nucleosome (histone octamer)
4. Amino-terminal tails of the core histones are accessible to proteases
5. Treatment of nucleosomes with limiting amounts of proteases that cleave after basic amino acids, specifically remove the amino-terminal “tails” leaving the histone core intact

Question 12

How do we know that DNA wraps around the histone core?

Answer 12

X-ray crystallography

Question 13

What type of supercoiling is expressed in the compacted DNA? By how much is the DNA compacted

Answer 13

Left-handed supercoil. 6-7 fold compaction.

Question 14

What is the histone-fold motif?

Answer 14

• A motif that has three alpha helices and two loops, contacting with DNA minor groove and phosphate backbone.
• Each two (H3-H4, H2A-H2B) forms head-to-tail heterodimer, which has a V-shaped structure and has 3 DNA binding sites which are unspecific but with some bias

Question 15

Which DNA sequences have high affinity in binding with histones?

Answer 15

AT rich regions in minor groove have preference in contacting the histone octamer. AT base pairs stagered at 10bp intervals helps the DNA bend at these sequences

Question 16

What are the point of histone tails?

Answer 16

They mediate inter-nucleosome connections. Also chromatin remodelling (structural changes in chromatin) relies on modification of histone tails affecting DNA replication, repair, and transcription

Question 17

What is the role of H1?

Answer 17

Addition of H1 leads to more compact nucleosomal DNA and so it promotes DNA condensation. Also has the ability to alter DNA entry and exit angles. Control of these angles leads to further compaction

Question 18

What is H1’s alternate name?

Answer 18

Linker histone