1. Chromosome Organization
September 20, 2017

2. Chromosome Organization
Prokaryotic and Eukaryotic cells have a DNA packaging problem
The DNA in one human diploid cell is about 2 meters long (6.5ft!).
It needs to squeeze into 15μm nucleus reducing its length as much as 10,000 fold
How does it do this?
DNA in prokaryotic and eukaryotic cells are organized into chromosomes. Chromosomes are defined as the DNA and its associated proteins

3. Chromosome Organization
Why is chromosome organization important?
Chromosomes are compact forms of DNA—allows DNA to fit inside a cell
Chromosomes stabilize DNA and protect it from damage
Allows for equal partitioning of DNA into daughter cells during cell division
Organizes the accessibility of other proteins to DNA. Critical for the regulation of DNA replication and transcription
Prokaryotic and Eukaryotic cells have different mechanisms for compacting DNA

4. DNA in cells Prokaryotic OR Eukaryotic?
Do not have membrane bound organelles; no nucleus
Linear chromosome
Have membrane bound organelles; have a nucleus
Circular chromosomes
Plasmid DNA is a small circular piece of DNA, often present in multiple copies. Typically not essential for growth.
Most cells are diploid (two copies of each chromosome), but cells can be haploid (one copy; gametes) or polyploid (many copies of each chromosome).

5. DNA in cells Prokaryotic Cells
Do not have membrane bound organelles; no nucleus
Circular chromosomes
Plasmid DNA is a small circular piece of DNA, often present in multiple copies. Typically not essential for growth.
Eukaryotic Cells
Have membrane bound organelles; have a nucleus
Linear chromosome
Most cells are diploid (two copies of each chromosome), but cells can be haploid (one copy; gametes) or polyploid (many copies of each chromosome).

6. Prokaryotic Chromosome Organization
Prokaryotic Chromosomes:
Prokaryotic circular DNA molecules are much larger than the bacteria cell (x1500)
Bacterial DNA is compacted and organized into a nucleoid. A nucleoid contains DNA, proteins and RNA

7. Prokaryotic Chromosome Organization
Prokaryotic Chromosomes:
Prokaryotic circular DNA molecules are much larger than the bacteria cell (x1500)
Bacterial DNA is compacted and organized into a nucleoid. A nucleoid contains DNA, proteins, and RNA
DNA is looped around anchoring proteins and loops are supercoiled

8. Eukaryotic Chromosomal Organization
Half the molecular mass of a eukaryotic chromosome is protein.
What are the majority of proteins?
Histones, Chromatin
Histones!

9. DNA Packaging

10. The majority of DNA is packaged into nucleosomes
Nucleosomes: Composed of a histone core and the core DNA.
Histones: Small positively charged proteins that associate with DNA. Form the protein “core” of a nucleosomes
Histone core: 2 copies of histones H2A, H2B, H3 and H4 (8 total!)
Core DNA: DNA that is most tightly associated with the nucleosome. Average length is 147bp
Linker DNA: DNA between each nucleosome. The size of linker DNA is highly variable.
Chromatin: The term given to describe DNA with its associated proteins.
Histones, Chromatin

11. The core DNA sequence is 147 bp?
Perform MNase Assay
MNase = Micrococcal Nuclease Assay
MNase cleaves DNA non-specifically
MNase cleaves protein- free DNA.
Isolate DNA from the protein
DNA band was 147bp

12. DNA Fragmentation in Apoptosis

13. DNA Fragmentation in Apoptosis

16. How is DNA packaged into nucleosomes?
Core Histones
Positively charged amino acids!
Linker Histones

17. Histone Protein Structure
Positive
The histone-fold domains of histones are conserved
Each core histone has an unstructured N-terminal tail

18. Nucleosomes Assembly
The histone core is composed of 2 copies of each core histone
H3 and H4 form dimers. H3-H4 dimers interact to form tetramers
H2A and H2B form dimers. H2A –H2B dimers interact to form tetramers.

19. Nucleosomes Assembly Nucleosomes are assembled in an ordered fashion!
Step 1: Two H3-H4 dimers form a tetramer
Step 2: H32:H42 tetramer binds to double stranded DNA
Step 3: Two H2A- H2B dimers bind are recruited to the DNA molecule forming a complete nucleosome
Step 1
Step 2
Step 3

20. How histones interact with DNA
There is a high affinity of histones for DNA
The DNA becomes distorted when bound to histones
DNA sequence specificity?

21. How histones interact with DNA
There is lack of DNA sequence specificity.
Histones form about 140 hydrogen bonds with the sugar-phosphate backbone and with the minor groove of DNA.
Why does binding to the sugar-phosphate backbone suggest a lack of DNA sequence specificity?
Answer: Histones form hydrogen bonds with the minor groove

22. Major and Minor Groove
In a double helix, one edge of the base pair is exposed in the minor groove and other edge is exposed in the major groove.
Therefore: The edges of each base pair are exposed in the major and minor grooves!
Minor and Major Groove:
Readings: Chapter

24. If a protein was making chemical bonds with the major groove, could a G-C pair be distinguished from a C-G pair?
Same question but with minor groove?

25. Nucleosomal Organization
How could you use the MNase assay to determine if DNA sequences that wrapped around a histones was similar?

26. Eukaryotic Chromosomal Organization
Nucleosomes and DNA Compaction
DNA wrapping around the histones (10nm) results in 6 fold compaction
Needs to be about 10,000 fold—depending on genome size
Nucleosomes link together to build a 30-nm fiber with help of Histone H1

27. Histone H1 binds to the linker DNA
10nm
30nm
Yes– It increase it from 147 to about 167.

28. Histone Tails are required to generate the 30nm fiber
Yes– It increase it from 147 to about 167.

31. True nature of the chromosome scaffold remains a mystery.
Further compaction
Histones alone are not the answer. The histone compaction (with Histone H1) results in about a 40 fold reduction. What does the rest?
True nature of the chromosome scaffold remains a mystery.