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SBC 275 last notes

Eukaryotic nucleus
-Eukaryotes have a true nucleus which is
surrounded by double layer membrane
...
g 46 in humans
...
9 A °, the structure consist of
147 base pairs (bp) of DNA wrapped in 1
...

-The core histones have ‘tail’ domains, which are long
disordered structures and play important roles in the
interactions between nucleosomes
...

-Chromatin is a negatively charged polymer that
produces electrostatic repulsion between
adjacent regions because only about half of the
DNA negative charges (from the phosphate
backbone) are neutralized by the basic core
histones
...

• Thus, the chromatin structure can be dynamically changed
depending on the electrostatic state of its environment
...

• In eukaryotic nucleus, the chromatin resembles a
disordered assembly of 10-nm fibers
...


• Because chromatin is composed of an irregular
and dynamic 10-nm fiber and does not have a
crystal-like long-range order, chromatin in the cell
is considered ‘liquid-like’ rather than static solidlike substance
...

• - The tail domains of histones H3 and H4 play
crucial roles in forming these various structures
...

• In more condensed, irreg-ular, or
interdigitated structures (e
...
, topologically
associating domains, TADs), the tails interact
with distant nucleosomes (long-range
interactions)
...


10

• Euchromatin and Heterochromatin
-are two distinct types of chromatin that are
revealed through microscopic observations of
interphase nuclei
...

- heterochromatin-like staining is often evident
at the nuclear periphery, suggesting that it is
enriched in this part of the nucleus
...

-The euchromatin stains less intense than
heterochromatin because heterochromatin has
tighter DNA packaging
...

- Heterochromatin is only found in eukaryotes but
euchromatin is found in both prokaryotes and
eukaryotes
...

12

-heterochromatin is responsible for gene regulation
and protection of chromosomal integrity
...


-In mammalian cells, heterochormatin are of two types:
constitutive and falcultative
...

- It is defined as the region of the chromosome that
remains condensed throughout the cell cycle
...


14

• Facultative heterochromatin is euchromatin
that will adopt heterochromatic properties in
a developmentally controlled manner,
suggesting temporal silencing of regions
of the genome
...

• It is a distinct heterochromatic structure that
accumulates during cellular senescence
...
Histones
1
...

the four “core” histones present as an
octameric unit in the nucleosomes
 are positively charged proteins, containing
relatively large amounts of lysine and arginine
17

bind directly to the DNA fiber by noncovalent
forces-, the electrostatic interactions between
positively
charged
residues
on
the
histonesand DNA phosphates
The C-terminal contains a ‘‘histone-fold’’ motif
that is shared by many proteins, including a
number of transcription factors and is involved
in
histone:histone
and
DNA:histone
interactions
...

They are mobile and flexible extending out of
the nucleosome core in a manner that frees
them for other interactions
...
Core Histone Variants (CHV)
-Chromatin can be remodeled by replacement of
major histones with specific histone variants
-Mostly occurred by gene duplication
-are often expressed at specific stages during
embryogenesis or during cell differentiation
...
g
...
Occurs in
mammalian kinetochores
...

• H2A
...

• –In euchromatin areas, H3 and H2A are replaced
by H3
...

• H2AX- represents 2–25% of H2A in a cell and is
phosphorylated on serine-139 at the site of
double-strand DNA breaks
...


21

• Linker Histones
-Linker histones (H1, H5) are highly dynamic &
located on the outside of the nucleosome
...

- are constructed from a highly conserved 79residue central globular domain with flexible
flanking N- and C-terminal domains
...

-It contains a conserved globular region and
extended amino- and carboxy-termini, the
latter being rich in lysines and able to interact
strongly with DNA
...
Sites of Lys/Arg residue m
ethylation and Ser phosphorylation are indicated
...
It consists of two copies of each of
the four core histone proteins
...
e
...

-In a symmetrical model for the nucleosome, the
H32- H42 tetramer provides a kernel for the
shape while the H2A-H2B dimers are in the
top and beneath positions i
...


27

28

-The diameter of the octamer is accounted for
by the H3 2 ·H4 2 tetramer
...

-All core histones have the structural motif of
the histone fold (three α-helices are
connected by two loops)
...
5 turns of the DNA
double helix (H2A-H2B binds at +3
...
5 - +3) (positions are numbered
from 0 to +7) through the phosphodiester
backbones (for easier packaging of DNA
irrespective of sequence)
...
e
...
Each histone also has a
flexible N-terminal tail, that extend out of the
nucleosome
...


33

•Histone modifications
-All the histones are modified by covalently
linking extra moieties to the free groups of
certain amino acids
...

-The modifications have important effects on
the structure of chromatin and in controlling
gene expression
...

-Histone
residues
modifications
:methylation,
phosphorylation,
acetylation,
sumolyation,
ubiquitinated and ADP-ribosylation
...
Each histone protein consists of the
N- and C-terminal tails and a central globular domain (gray
box)
...


Histone acetylation
- Histone acetylation occurs by the enzymatic addition
of an acetyl group (COCH3) from acetyl coenzyme A
...

-Occurs in lysine residues
-highly dynamic and regulated by the opposing action
of
two
families
of
enzymes,
histone
acetyltransferases (HATs) and histone deacetylases
37
(HDACs)
...

-Acetylation neutralize the lysine’s positive charge and
this action has the potential to weaken the interactions
between histones and DNA
- Histone H3 acetylation may be increased by inhibition
of HDACs and decreased by HAT inhibition
...
An imbalance in the equilibrium of histone
acetylation has been associated with tumorigenesis
and cancer progression
...

-It takes place on serines, threonines and tyrosines,
predominantly, but not exclusively, in the N-terminal
histone tails
...

-Histone kinases transfer a phosphate group from ATP to
the hydroxyl group of the target amino-acid side chain
...

-It does not alter the charge of the histone protein
...

-Lysine is methylated at N terminal tail by histone lysine
methyltransferase ((HKMT) e
...

-Dot1 enzyme that methylates H3K79 within
the histone globular core and does not contain a SET
domain
...

-The type-I enzymes generate Rme1 and
Rme2as, whereas the type-II enzymes
generate Rme1 and Rme2s
...

43

-PRMTs enzymes transfer a methyl group from S
adenosyl methionine (SAM) to the ωguanidino group of arginine within a variety of
substrates
...
g
...
It
utilizes FAD (flavin adenine dinucleotide) as
co-factor
...

- In mammalian cells, this reaction on histones
is catalysed by the peptidyl deiminase PADI4, which
converts peptidyl arginines to citrulline
...

-PADI4 also converts mono-methyl arginine to
citrulline, thereby effectively functioning as an
arginine demethylase
...

-Such reactions occurs in histones also
...

- Histones H2A, H2B and H4 have been shown to
be modified by O-GlcNAc
46

ADP ribosylation
- Ribosylation is the addition of one or more ADP ribose
moieties to a protein
-Histones are mono- and poly-ADP ribosylated
on glutamate and arginine residues
...

-Poly-ADP ribosylation of histones is performed by the polyADPribose polymerase (PARP) family of enzymes and
reversed by the poly-ADP-ribose-glycohydrolase family of
enzymes
...

-These modifications significantly increase upon DNA
damage

48

Ubiquitylation
- Ubiquitin itself is a 76-amino acid polypeptide that is
attached to histone lysines via the sequential action of
three enzymes, E1- activating, E2-conjugating and E3ligating enzymes
...
e
...
e
...

-For histones, mono-ubiquitylation is the most relevant
...
g- Ubiquitylation of histones H2A and H2B yields
H2AK119ub1 which is involved in gene silencing and
H2BK123ub1 plays an important role in transcriptional
initiation and elongation respectively
...

Sumoylation
- Sumoylation involves the covalent attachment of small
ubiquitin- like modifier molecules to histone lysines via
the action of E1, E2 and E3 enzymes
...

-This modification has mainly been associated with
repressive functions
...

- It exists in yeast and mammals (mouse) where the
first 21 amino acids of H3 are removed
...


51

•Non histone proteins associated with
DNA in eukaryotic nucleus
-In addition to the core histones, the DNA scafford
contain several proteins: large amounts of
histone Hl (located in the interior of the fiber)
and topoisomerase II

The presence of topoisomerase II unwinds DNA
during DNA replication
...

-Several drugs used in cancer chemotherapy are
topoisomerase II inhibitors that allow the
enzyme to promote strand breakage but not
the resealing of the breaks
...

-The primary structure of SMC proteins consists of
five distinct domain (see fig below (a))
...


-The dimeric proteins forming a V-shaped
complex are tied together through the
protein's hinge domains where one N and one
C domain come together to form a complete
ATP-hydrolytic site at each free end of the V
(see fig
...


-Proteins in the SMC family occur in all types of
Organisms from bacteria to humans
...
This
linkage is essential if chromosomes are to
segregate properly at cell division
...

-The cohesins and condensins are essential in
orchestrating many changes in chromosome
structure during the eukaryotic cell cycle

B
...
Nucleosomes
- a structural component of the chromatin fiber,
and a modulator of gene expression
...
The positioning of
nucleosomes can be modulated by minor
alterations in the DNA sequence and by DNA
methylation
...

-It comprises of nucleosome
...
The protein
components are histones
...


63

-Nucleosomes are an invariant component of
euchromatin and heterochromatin in the
interphase nucleus, and
of mitotic
chromosomes
...

-When chromatin is replicated, the nucleosomes
must be reproduced on both daughter duplex
molecules
...
Approximately half of
the protein mass is the nucleosomes
...


66

-There is also
nonhistones
proteins of
chromatin but in lower amounts than the
histones
...

-Individual nucleosomes can be obtained by
treating chromatin with the endonuclease
micrococcal nuclease
...

68

-The nucleosome contains ~200 bp of DNA
associated with a histone octamer that
consists of two copies of the core histonesH2A, H2B, H3, and H4
...

-The types of H2A and H2B occur in all
eukaryotes, but show species-specific
variation in sequence
...
H1 role is different from
the core histones
...


72

-The shape of the nucleosome shape is like a
flat disk or cylinder, of 11nm diameter and a
height of 6 nm
...
The DNA
follows a symmetrical path around the
octamer
...

73

74

-one DNA turn around the nucleosome takes
~80 bp of DNA
...
This cleaves
the DNA into integral multiples of a unit
length
...

76

77

- The ladder is generated by groups of
nucleosomes
...

- When nucleosomes are fractionated on a
sucrose gradient, they give a series of discrete
peaks that correspond to monomers, dimers,
trimers, tetramers etc
...

-The monomeric nucleosome contains DNA
eqivalent of the unit length e
...
the
nucleosome dimer contains DNA of twice the
unit length
...
In conclusion, the
existence of the 200 bp ladder in any
chromatin indicates that the DNA is organized
into nucleosomes
...


81

->95% of the DNA of chromatin can be
recovered in the form of the 200 bp ladder
...

-In their natural state, nucleosomes are closely
packed, with DNA passing directly from one to
the next
...

82

-The length of DNA per nucleosome varies for
individual tissues in a range from 154 (in
fungus)-260 bp (in sea urchin sperm)
...

-The association of DNA with the histone
octamer forms a core particle
...

84

- The core particle DNA is relatively resistant to
digestion by nucleases
...
Its length varies from as little as 8 bp to
as much as 114 bp per nucleosome
...


85

-The 8-114bp regions are susceptible to early
cleavage by the enzyme
...

-Micrococcal nuclease initially cleaves between
nucleosomes
...

-End-trimming reduces the length of DNA first to
~165 bp, and then generates core particles
with 146 bp
...
The H1 is lost during the
degradation of nucleosome monomers
...
This suggests that
H1 could be located in the region of the linker
DNA immediately adjacent to the core DNA
...

-Within the nucleosome, DNAase I and DNAase
II enzymes make single-strand nicks in DNA
where they cleave a bond in one strand, but
the other strand remains intact at this point
...

- Using radioactively end-labelling of DNA,
denaturation and electrophoresing the
resultant fragments, a ladder is generated i
...

-The interval between successive steps on the
ladder is 10-11 bases
...
The cleavage
sites are numbered as S1 through S13 (where
S1 is ~10 bases from the labeled 5 ′ end, S2 is
~20 bases from it, and so on)
...

-The cutting periodicity (the spacing between
cleavage points) coincides with the structural
periodicity (the number of base pairs per turn
of the double helix) i
...
the distance between
the sites corresponds to the number of base
pairs per turn
...
g
...
5 bp/turn
...
Thus, a cutting site
represents a short stretch of bonds on both
strands, exposed to nuclease action over 3-4
base pairs
...
0 at the ends, 10
...
The DNA
has more bp/turn than its solution value in
the middle, but has fewer bp/turn at the ends
...
17 bp/turn, and for DNA in solution is 10
...

-The crystal structure of the core particle suggests
that DNA is organized as a flat superhelix, with
1
...

-Regions of high curvature occur at positions ± 1
and ± 4; corresponding to S6 and S8, and to S3
and S11, which are the sites least sensitive to
DNAase I
...
Sites
may be numbered S1
to S13 from one end;
or taking S7 to identify
coordinate 0 of the
dyad symmetry, they
may be numbered -7
to +7
...
59 left-handed superhelical turns
with a diameter of 80
...

97

- The central 129 bp are in the form of B-DNA,
but has a substantial curvature that is
needed to form the superhelix
...
These
conformational changes explains why the
central part of nucleosomal DNA is not usually
a target for binding by regulatory proteins (
regulatory proteins typically bind to the
terminal parts of the core DNA or to the linker
sequences)
...
The
alteration is confined to the immediate
vicinity of the replication fork, but the
nucleosomes reform more or less immediately
behind the fork as it moves along
...
e
the assembly of nucleosomes is directly linked
to the replisome that is replicating DNA
...
A preformed octamer binds to DNA i
...
This relates to
what happens in vivo as it reflects the capacity of
chromatin to be remodeled by moving histone
octamers along DNA
...
An indirect interaction where a tetramer of
H32 ·H42 binds first, then two H2A·H2B dimers
are added
...
i
...


102

In vitro two paths are predicted

103

-- Accessory proteins are involved in assisting
histones to associate with DNA where they act
as "molecular chaperones" that bind to the
histones releasing either individual histones or
complexes ( H3 2·H42 or H2A·H2B) to the DNA in
a controlled manner
...
It requires a chromatin
assembly factor (CAF-1) an ancillary factor,
CAF-1, that consists of >5 subunits, with a
total mass of 238 kD
...
This provides the link between
replication and nucleosome assembly,
ensuring that nucleosomes are assembled as
soon as DNA has been replicated
...

-During replication, the histone octamers are
not conserved i
...

-During replication, H2A·H2B dimers and
H3 ·H4 tetramers are conserved i
...

106

-Replication fork passage displaces histone
octamers from DNA
...
Newly
synthesized histones are assembled into H32H42 tetramers and H2A-H2B dimers
...

-In eukaryotic cells, during S phase sufficient
histone proteins to package an entire genome
are synthesised for the duplication of
chromatin
...
e the same quantity of histones
must be synthesized that are already
contained in nucleosomes
...

- The pathway for assembling chromatin from
this equal mix of old and new histones during
S phase is called the replication-coupled (RC)
pathway
...
This is necessitated
by damage to DNA or nucleosomes displaced
during transcription
...

112

-H3
...
It differs from the
highly conserved H3 histone at 4 amino acid
positions
...

-Assembly of nucleosomes containing an
alternative to H3 also occurs at centromeres
...

113

-Using the nucleosomal digest, each eukaryotic
chromosome contains many replicons
...
This
occurs by the replication-independent
assembly pathway because the replicationcoupled pathway is inhibited for a brief period
of time while centromeric DNA replicates

114

-The replication fork displaces histone octamers,
which then dissociate into H3 2·H42 tetramers
and H2A·H2B dimers
...

-Nucleosomes assemble ~600 bp behind the
replication fork
...
This is followed
by binding of the two dimers (H2A·H2) to each
tetramer to complete the histone octamer
...

116

Nucleosome phasing/ positioning
-Nucleosome positioning is defined as the
placement of nucleosomes at defined sequences
of DNA instead of at random locations with
regards to sequence
...


117

• Indirect end labeling/ DNA foot printing
-It is a technique for examining the organization
of DNA by making a cut at a specific site and
isolating all fragments containing the
sequence adjacent to one side of the cut
...


118

-The DNA is isolated
...
The
unprotected
or susceptible regions are
cleaved by non-specific endonucleases This
results in the removal of the protecting
protein
...

119

-The fragment is cleaved with a restriction
enzyme that has only one target site in the
fragment thus it cuts at a unique point
producing two fragments, each of unique size
...

-A probe representing the sequence on one side
of the restriction site is used to identify the
corresponding fragment in the double digest
...

-The identification of a single sharp band
demonstrates that the position of the
restriction site is uniquely defined with respect
to the end of the nucleosomal DNA

121

122

In the absence of
nucleosome
positioning, a
restriction site lies at
all possible locations in
different copies of
the
genome
...


123

-the existence of a specific band in the indirect
end-labeling technique represents the distance
from the restriction cut to a preferred
micrococcal nuclease cleavage site
...

-Nucleosome positioning is both intrinsic and
extrinsic
...

-It is extrinsic because the first nucleosome in a
region is preferentially assembled at a particular
site
...

125

-Thus, the deposition of histone octamers on
DNA is intrinsic
...

• Structural features of DNA affect placement of
histone octamers
...
A·T-rich regions are arranged in such a way
that the minor groove faces in towards the
octamer
...
G·C-rich regions are arranged so that the
minor groove points out
...
Long runs of dA·dT (>8 bp) are not
positioned in the central superhelical turn of
the core
...
Sequences that cause DNA to take up more
extreme structures may have effects such as
the exclusion of nucleosomes, and thus could
cause boundary effects
...
This results in the
location proceeding away from the first
defined nucleosome at the boundary
...


128

-The location of DNA on nucleosomes can be
described in two ways:
a)
...

129

-It describes the linear position of DNA relative
to the histone octamer
...


130

b) rotational positioning- describes the location
of the histone octamer relative to turns of the
double helix, which determines which face of
DNA is exposed on the nucleosome surface
...

-Any movement that differs from the helical
repeat (~10
...

131

-Nucleotides on the inside are more protected
against nucleases than nucleotides on the
outside
...
Shifting the DNA by
10 bp brings the next turn into a linker region
...
g to the
micrococcal nuclease)
...
E
...
The positioning involving the specific
placement of nucleosomes at promoters
...
Some regulatory factors can bind to DNA
only if a nucleosome is excluded to make the
DNA freely accessible, and this creates a
boundary for translational positioning
...


135



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