Prof' Giovanni Bosco

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Prof. Giovanni Bosco

• Thurs (9/21) Tues (9/26) Lecture Slides

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Suppose you are going on a long trip. Some parts
are predictable and other parts are not. You
are only allowed one average size suitcase.How
would you pack for this trip?
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The DNA packaging problem1.fitting it all into
the nucleus.2.keeping it accessible.3.need to
unpack and re-pack without too much trouble.
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The DNA packaging problem1.fitting it all into
the nucleus.(How do you pack for a long
tripwhen you have limited space?)
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If each nucleotide pair were 1mm
then the human genome would extend 2,000
miles when stretched end to end (3,200Km).
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The billions of basepairs (3,200Km) must fit into
a tiny nucleus! The average nucleus is about 5um
in diameter. Must fit 3.2x103 meter long DNA into
5x10-6 meter nucleus. And so...?
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The billions of basepairs (3,200Km) must fit into
a tiny nucleus! The average nucleus is about 5um
in diameter. Must fit 3.2x103 meter long DNA into
5x10-6 meter nucleus. That means that DNA in one
nucleus is 109 (a billion) times longer then the
diameter of a nucleus.
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That means that DNA in one nucleus is 109 (a
billion) times longer then the diameter of a
nucleus. So why do we care? Why is this
important to think about?
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Inheritance of traits depends on proper
transmission of GENES. Genes reside on
chromosomes. Chromosomes are duplicated so that
each cell at cell division inherits an equal
amount of the genetic material.
unpack and re-pack every cell
division. (unpack repack after every stop on
your trip without losing anything)
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unpack and re-pack every cell
division. (unpack repack after every stop on
your trip without losing anything)
Of the 30,000 genes in each of our cells almost
every single gene is indispensable at some point
during the life of that cell. Must not lose or
damage any genes!
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How is DNA packaged into such a small space while
remaining accessible?(Does you no good to pack
reallywell if you cannot actually get atand use
the stuff you packed.)
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AnswerChromatin

• Chromatin is DNA bound by proteins.

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The first order packaging of chromatin beads on
a string.
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Four important core histone proteins come
together to form an octamer (eight sub-units, two
of each). Histones are the major protein (but
not the only) component of chromatin.
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What about the structure of DNA presents a
challenge to the packaging problem?
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Histones are positively charged proteins.DNA is
negatively charged.What kind of interaction
would you predict histones make with DNA?
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Histones are positively charged proteins.DNA is
negatively charged.Histones have ionic
interactions with DNA (mostly)?
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The garden hose model. DNA wraps twice around
the octamer.
DNA
Histones
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The nucleosome is the basic, first order
packaging of DNA in the nucleus of the cell.
DNA double helix wraps twice around a single
histone octamer to form one nucleosome. The
DNA in between each nucleosome is called linker
DNA.
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How do we know what a nucleosome consists of?
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The experiment that first showed us how DNA was
bound in a nucleosome. Chromatin (DNAprotein)
was digested with nuclease. This chewed up the
linker DNA. (Why does some DNA remain?) Then
high salt concentration, for example Na, was
used to dissociate the DNA from proteins.
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DNA was separated from proteins. DNA and
proteins were analyzed separately.
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DNA was separated from proteins. DNA and
proteins were analyzed separately. It was found
that most DNA fragments averaged 146Bp in
length. It was found that for every DNA molecule
there were two molecules of each of the four
histones.
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Questions?
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Histones share a common structure, the histone
fold, where most of the contacts with DNA are
made.
The histone fold is also important for different
histone molecules to come together and
form dimers.
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How an octamer is formed.
First, H3 H4 form one dimer, and H2A H2B form
another dimer.
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Then, two H3/H4 dimers form a tetramer, and two
H2B/H2A dimers from a tetramer. The two
tetramers come together to form an octamer.
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The octmaer binds to DNA very strongly and bends
the DNA around it.
The energy of ATP and special chromatin enzymes
are required to assemble histones onto the DNA
double helix.
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Questions?
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9/26 Lecture Starts Here
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One full turn of the double helix takes
10.4 basepairs.
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The octmaer binds to DNA very strongly and bends
the DNA around it.
What might you predict about how A-Ts and G-Cs
are distributed on the DNA?
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The octmaer binds to DNA very strongly and bends
the DNA around it.
What might you predict about how A-Ts and G-Cs
are distributed on the DNA? One might expect
that a T-A occurs every turn of the helix (10
basepairs) and G-C also occurs every turn of the
helix. AND, T-A must be facing the opposite way
as do G-C. So, T-A pairs are separated from
G-C by about 5 basepairs.
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So, T-A pairs are separated from G-C by about 5
basepairs. Why might this be important???
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If your name last name starts with A-H Fold
your paper onto itself the same direction every
time.I-Z Fold your paper onto itself changing
sides after every fold.
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Now gently unfold your paper and do NOT flatten
it out.What do you find?
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What if the distance between adjacent nucleotide
on the same strand was not always exactly 0.34nm??
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G-C
T-A
G-C
T-A
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So, T-A pairs are separated from G-C by about 5
basepairs. Why might this be important??? The
sequence of the DNA and the spacing of that DNA
can contribute to the curvature and strength
of the interaction between the DNA and the
histones.
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The DNA packaging problem1.fitting it all into
the nucleus.2.keeping it accessible.3.need to
unpack and re-pack without too much trouble.
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The second order packaging nucleosomes fold
onto themselves.
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DNA binding proteins (like transcription factors
and DNA replication proteins) can remain bound
to DNA as the nucleosomes fold onto themselves.
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Remodeling enzymes are required to
allow non-histone proteins to assemble onto DNA.
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Histone N-terminal tails do not make contacts
with DNA but are extremely important for histone
function. Each tail is unique to each type of
histone.
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The histone fold of the proteins are on the
interior and surface of the core nucleosome. The
N-terminal tails stick out of the core nucleosome.
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Histone tails stick out of the
nucleosome. These tails are targets for enzymes
that can modify the amino acids of these histone
tails. These modifications are reversible.
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A tremendous number of chemical modifications of
the histone tails of the nucleosome can produce a
tremendous amount of variability.
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The variability in histone tail modifications can
have profound effects of gene expressions and
chromosome structure.
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These tails are targets for enzymes that can
modify the amino acids of these histone
tails. How do you imagine modification of
histone tails might change the way DNA is
packaged into a nucleosome?
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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Changes the charge
• of the histone protein(s)
• within that nucleosome.

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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Changes the charge
• of the histone protein(s)
• within that nucleosome.
• Recall that negative DNA
• and positive histones have
• strong ionic (charge based)
• interactions.

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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Changes the charge
• of the histone protein(s)
• within that nucleosome.
• For example Acetylation
• of histones makes them
• more negatively (less
• positively) charged, thus
• weakening their hold on
• DNA.

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Questions?
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When histones have a weakened hold on DNA,
then DNA binding proteins (like transcription
factors and DNA replication proteins) can remain
bound to DNA or compete with histones and
initiate binding.
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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Specific modifications
• on histones could create
• binding sites for other,
• non-histone proteins.

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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Specific modifications
• on histones could create
• binding sites for other,
• non-histone proteins.
• Non-histone proteins
• could use energy from ATP,
• for example, to move
• histones off or away from
• important DNA sites.

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• 2. Specific modifications
• on histones could create
• binding sites for other,
• non-histone proteins.
• Non-histone proteins
• could use energy from ATP,
• for example, to move
• histones off or away from
• important DNA sites.

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• How do you imagine
• modification of histone
• tails might change the
• way DNA is packaged
• into a nucleosome?
• Keep in mind that
• changing the charge of
• histone proteins and/or
• allowing other proteins to
• load onto the DNA could
• result in tighter packaging
• of DNA.

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The DNA packaging problem3.need to unpack and
re-pack without too much trouble.
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The DNA packaging problemSo, one huge
advantage of histone modifications is that they
are reversible. This allows the cell to unpack
and re-pack small regions of DNA without too much
trouble.
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Example of a reversible modification
Histone H3 phosphorylated in mitosis.
Histone H3 de-phosphorylated in non-mitotic
cells. Every eukaryotic cell on the planet does
this. We still do not understand the
significance of this histone modification.
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The DNA packaging problem3.need to unpack and
re-pack without too much trouble.Compartments
make it easierto organize stuff. How?
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What are some examples of compartments within the
nucleus?
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Within the nucleus various COMPARTMENTS
hold/store genes that are similar or function at
the same time. For example, the NUCLEOLUS is a
nuclear COMPARTMENT that holds almost all the
genes that function to make ribosomes. rDNA,
tRNA and ribosomal protein genes cluster in the
NUCLEOLUS.
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Within the nucleus various COMPARTMENTS
hold/store genes that are similar or function at
the same time. This allows for efficient and
coordinated one stop shopping when cells need
to ramp up or ramp down ribosome synthesis.
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The nuclear lamina and inner membrane can be
thought of as a compartment. Telomeres
(chromosome ends), centromeres and a variety of
genes can be localized to the nuclear
lamina/membrane.
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Genes localized to the nuclear lamina/membrane
are usually inaccessible for transcription (gene
activation).
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Keeping DNA accessible. The way it is packaged
allows for loops or spooling out of tiny
regions as they are required for gene expression
or as they are replicated.
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Keeping DNA accessible. The way it is packaged
allows for loops or spooling out of tiny
regions as they are required for gene expression
or as they are replicated.
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We imagine that such loops and spools represent
regions of the chromosomes with more or
less gene activity. These structures are dynamic
and can differ dramatically from one cell type
to another.
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The DNA packaging problem1.fitting it all into
the nucleus.2.keeping it accessible.3.need to
unpack and re-pack without too much trouble.
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Questions?
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