The document discusses DNA packaging in eukaryotic cells. It describes how DNA is wrapped around histone proteins to form nucleosomes, which are regularly spaced beads on a string of chromatin. Nucleosomes further condense into a 30nm fiber through interactions between nucleosomes. Higher-order folding involves loops of fiber and coiling to achieve the extreme compaction needed to fit a cell's full genome inside the nucleus.

1. DNA Packaging

2. • Lecture Overview
• Structure of chromosomes and chromatin.
• DNA packing and histone modification.

3. Objectives:
• Understand the nature of chromatin - how DNA and histones
interact to form nucleosomes.
• Know the basic structures of the 10 nm chromatin fiber and the
30 nm chromatin fiber.
• Realize that chromosomes are single pieces of DNA packaged
into chromatin.

4. Genetic Material in the Living Cells
• Cells contain a nucleus surrounded by a nuclear
membrane in eukaryotic cells, and a nuclear region in
the prokaryotic cells.
• In a non-dividing cell the nucleus is filled with a
thread-like material known as "chromatin".
• Chromatin is made up of DNA and proteins (mainly
histones and some non-histone acidic proteins).

5. The Normal Human Chromosomes
• Normal human cells contain 23 pairs of homologous
chromosomes:
i. 22 pairs of autosomes.
ii. 1 pair of sex chromosomes.
• Autosomes are the same in males and females
• Sex chromosomes are:
i. XX in females
ii. XY in males.
• Both X are homologous. Y is much smaller than X and has only a
few genes.

6. Chromosomes
• One member of each chromosome pair is derived from each
parent.
• Somatic cells have diploid complement of chromosomes i.e. 46.
• Germ cells (Gametes: sperm and ova) have haploid
complement i.e 23.
• Individual chromsomes are recognized by
i. arm lengths ( p- short, q -long).
ii. centromere position (metacentric, sub-metacentric, acrocentric,
telocentric).

7. Composition of Chromosome
DNA
Histones
(Major proteins)
Non-histone
(Small amounts)
p
q
 The chromosomes themselves are macromolecular entities that must be
synthesized, packaged, protected, and properly distributed to daughter cells
at cell division.
 Significant segments of every chromosome are dedicated to these
functions.

8. DNA Condensation: Why?

9. A severe problem of packaging
1. Largest human chromosome: ~3 x 108 bp
2. A typical cell = 10 μm = 10 x 10-6 m
3. Therefore the DNA must be compacted ~104-fold.
This is like fitting an 11-mile-long string into a 6-foot box
How
long is
it?

10. Higher-order DNA compaction in a eukaryotic chromosome. This model shows the levels of
organization that could provide the observed degree of DNA compaction in the chromosomes of
eukaryotes. First the DNA is wrapped around histone octamers, then H1 stimulates formation of the
30 nm filament. Further levels of organization are not well understood but seem to involve further
coiling and loops in the form of rosettes, which also coil into thicker structures. Overall, progressive
levels of organization take the form of coils upon coils upon coils. It should be noted that in cells, the
higher-order structures (above the 30 nm filament) are unlikely to be as uniform as depicted here.
DNA packing- Superstructure

11. • Eukaryotes contain thousands of times more DNA than do
bacteria, and as a result, the DNA–condensation problems
of eukaryotes—compacting the DNA so that it fits in the
cell nucleus—are more complex than those of bacteria.
• Bacteria do not contain nucleosomes, although they have
small, basic (positively charged) proteins that are involved
in condensing their DNA.

12. Continue..
• DNA compaction must be dynamic, because changes in the
degree of condensation must occur quickly and when
needed, as the cell passes through the stages of the cell
cycle.
• Furthermore, when in its most highly compacted form,
DNA is not accessible to transcription or replication
enzymes, so it must be able to rapidly expose regions
containing genes that are required at any given moment,
and then condense again.
• Modification enzymes that alter the state of DNA
condensation, and can target their activity to specific
regions of the chromosome that must be transcribed or
replicated.

13. Nucleosomes: The Basic Units of DNA
Condensation
• The material of chromosomes, both protein
and DNA, is often referred to as chromatin.
The protein component is about equal in
mass to the DNA component.
• Histones constitute the largest protein
component of chromatin, are highly
conserved, basic proteins that assemble
into octameric complexes containing two
each of four different histone subunits.
• DNA wraps around the histones to form
condensed nucleosomes.

14. Nucleosome (10 nm diameter):
• 8 histones in bead & 1 outside.
• Each bead: is surrounded by 140 bpDNA and there are 60 bp in the linker region.
• Space between beads is about 14 nm.

15. Continue..
• Histones are rich in the basic amino acids arginine and
lysine, which together make up about 25% of the amino
acid residues in any given histone protein.
• Histone proteins are highly conserved among
eukaryotic cells.
• Histones H3 and H4 are nearly identical in all
eukaryotes, suggesting strict conservation of their
functions.
• Histones H1, H2A, and H2B show less sequence
similarity, but on the whole, they are more conserved
than other types of proteins.
• Salt bridges between positively charged histones and
negatively charges DNA play a major role in stabilizing
DNA-histone complex

16. Histone Octamers Organize DNA into
Repeating Units
• The first evidence that DNA is packaged into regularly organized units
came from studies in which chromosomal DNA was treated with a
nonspecific DNA nuclease, that cuts DNA wherever it is not associated
with proteins.
• The digested DNA fragments were then analyzed for size in an agarose
gel.
• If DNA is packaged by proteins into units of a particular size, the
nuclease would cleave the DNA between these units, and the protected
DNA segments would migrate in the gel as a ladder of unit-sized bands.
• The results of such experiments revealed a series of regularly spaced
DNA bands about 200 bp apart, indicating that DNA is packaged by
proteins into units that encompass approximately 200 bp

17. The earliest evidence of DNA packaging.
 Isolated chromatin was treated with nuclease and analyzed by agarose gel
electrophoresis.
 The result was a DNA ladder of fragments that differed in length by 200 bp,
suggesting that DNA packaging involves a repeat unit of 200 bp.
[Source: Roger Kornberg, MRC Laboratory of Molecular Biology.]

18. Histone representation in nucleosomes.
Histones within nucleosomes were separated by SDS–polyacrylamide gel
electrophoresis.
 Measurement of the band intensity showed histones H2A, H2B, H3, and H4 present
in equal stoichiometry, and histone H1 at about half the level of the other histones.
[Source: S. Panyim and R. Chalkley, Archiv. Biochem. Biophys. 130:337–346, 1969.]
Histones- main packing proteins.
• Consist of 5 classes: H1, H2A, H2B, H3, H4.
• When the protein-DNA units (nucleosomes) were
examined by SDS−polyacrylamide gel electrophoresis
(SDS-PAGE), four histone proteins (H2A, H2B, H3, and
H4) were found in approximately equimolar ratios .
• A fifth histone (H1) was present in about half the amount
relative to the other four histones. H1 is Lys rich.
• The five histones have molecular weights (Mr) between
11,000 and 21,000.

19. Characteristics of Histones

20. Chromatin Modifications Functions Regulated
Acetylation
Transcription, Repair, Replication,
Condensation
Methylation (lysines) Transcription, Repair
Methylation (arginines) Transcription
Phosphorylation Transcription, Repair, Condensation
Ubiquitylation Transcription, Repair
Sumoylation Transcription
ADP ribosylation Transcription
Deimination Transcription
Proline Isomerization Transcription
Types of histone modifications
Post-translational modifications on histone proteins alter chromatin
structure and, consequently, chromatin function

21. Nucleosomes
• Kornberg suggested that most of the 200 bp of DNA in a protein-DNA unit
is wrapped around a histone octamer composed of two copies each of
histones H2A, H2B, H3, and H4. These four histones have come to be
known as the core histones.
• The remainder of the DNA serves as a linker between nucleosomes, to
which histone H1 binds.
• Under physiological conditions, formation of the histone octamer from
individual histone proteins requires the presence of DNA.
Nucleosomes as beads on a string.
Regularly spaced nucleosomes consist of
core histone proteins bound to DNA.

22. Histone H1 Binds the Nucleosome to Form the
Chromatosome
• The histone octamer and associated DNA that form the nucleosome combine
with histone H1 to form the chromatosome.
• The addition of H1 to a nucleosome results in protection of an additional 20 to
22 bp of linker DNA adjacent to the nucleosome, and thus H1 is often referred
to as the linker histone.
• Only one H1 subunit is present per chromatosome, unlike the core histones,
which are present in two copies each.
• DNA binding in H1 is intrinsic to the central globular region, which contains
two DNA-binding sites.
• H1 binds only one of the linker DNA strands, and the second DNA site in
histone H1 binds to the central region of the DNA supercoil in the nucleosome
The binding of DNA by histone H1.
H1 has two DNA-binding sites, through which it makes
contact with one arm of linker DNA and the central
region of the DNA wrapped around the histone octamer.

23. Chromosomes Condense into a Compact
Chromatin Filament
• Nucleosomes condense into a compact filament with a width of about
30 nm, referred to as the 30 nm filament.
• H1 promotes condensation into the 30 nm filament, but it is not
essential for forming the filament.
• In contrast, the N-terminal tails of the core histones are absolutely
required, suggesting that the tails provide important nucleosome-
nucleosome contacts needed for 30 nm filament formation.
• There are two most widely accepted models for nucleosome
arrangement in the 30 nm filament:
i. Solenoid model
ii. Zigzag model

24. The 30 nm filament, a higher-order organization of nucleosomes.
The compact filament is formed by the tight packing of nucleosomes. Two proposed
models of filament structure are (A) the solenoid model and (B) the zigzag model.
[Source: (a) Barbara Hamkalo, Department of Molecular Biology and Biochemistry University of California, Irvine.]

25. Higher-Order Chromosome Structure Involves
Loops and Coils
• Inside chromosomes, DNA is much more highly
condensed than in the 30 nm filament.
• 30 nm filaments is appear to be organized in loops
estimated at 40 to 100 kbp long.
• chromosomal scaffold: Proteinaceous residue
after extraction of histones from chromosomes,
comprised mainly of Structural maintenance of
chromosomes (SMC) proteins.
• Regions of the DNA interact with chromosomal
scaffold proteins to give a protein core with DNA
loops sticking out of it.
• This protein core then coils up to further package
the DNA into the chromatids that are visible by
light microscopy in metaphase.

26. Summary
• Chromatin is the sum of the protein and nucleic acid that
comprise the material of chromosomes.
• The basic unit of DNA packaging is the nucleosome, composed of
about 200 bp of DNA wrapped around a histone octamer.
• The histone octamer contains a tetramer of histones H3-H4 and
two dimers of histones H2A-H2B. The amino acid sequences of
the histones are highly conserved among eukaryotes.
• Nucleosomes are linked together by DNA with one bound
molecule of H1.

27. Level of folding Consists of Base pair/turn
DNA double helix Nucleotides 10
Nucleosomes 200 bp each 100
30 Nanometer fiber 6 Nucleosomes /turn 1,200
Loops 50 Solenoids/loop 60,000
Miniband 18 loops 1,080,000
Chromatid 1,000,000 minibands
Summary of Chromosome Folding