Biochemistry 441 Lecture 14 March 10, 2000 Ted Young

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• Know then thyself, presume not God to scan.
• The proper study of mankind is man.
• Placed on this isthmus of a middle state,
• A being darkly wise and rudely great.
• -Alexander Pope from Essay on Man

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Biochemistry 441Lecture 18February 20, 2009Ted
Young

• Chromosome structure DNA condensation in
  chromosomes
• A typical human cell has 2 meters of DNA in a
  nucleus with a diameter of 10-5 meters. How does
  the DNA get packed into it? (Is there enough
  space? Do the calculation!)

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Chromosomes change structure during the cell cycle
This cohesion is what ensures that one
parents contributions are separated in the
daughter cells

• .

Cohesins, condensins, and separase are conserved
enzymes found in all eukaryotes. They are
essential for progression of the cell
cycle. Note that only one molecule is shown but
in a diploid cell there would be two identical
chromosomes (DNA molecules).
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Disrupted nucleus shows extended DNA fibers
NB ends are never seen in these Ems gt
chromosomes are made up of a single DNA molecule.
Fig 24-31
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Metaphase chromatin is densely packed

• Paired human chromosomes at metaphase. Note the
  diameter of fibrils-much thicker than a single
  DNA molecule.

Constriction at centromere always at the same
position of each chromosome
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Histones
Histone Residues Arg Lys Evolutionary
stability (UEP) H1 215 1 29 8 H2A 129
9 11 60 H2B 125 6 16 60 H3 135 13 10 330 H4
102 14 11 600 UEP unit evolutionary period
the time in megayears for a proteins amino acid
sequence to change by 1 after two species
diverge.

• Conclusions Histone amino acid sequences are
  remarkable stable. Implication the sequences
  have been conserved for functional reasons only
  this amino acid sequence will work.

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Calf thymus histone H4

• Red Arg/Lys underlined post-translationally
  modified by acetylation or methylation of Lys or
  Arg, phosphorylation of Ser, and ubiquitylation
  of lysine. Most modified amino acids are in the
  amino terminal 20 residues. What effect on
  histone charge would these modifications have?
• Pea Lys77gtArg Val60gtIle only two changes,
  implying strong selection for maintaining the
  exact amino acid sequence.

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Structural conservation of histones

• Each histone has a very similar structure (but a
  very different sequence) a long central a-helix
  flanked on each end by a turn and two a- shorter
  helices. Many other transcription-related
  proteins have a very similar structure and may
  have evolved from the same ancestral gene.

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The histone code hypothesis

• Modifications of histones convey epigenetic
  information.
• Epigenetic genetic alterations that are not
  encoded in the DNA.
• For example GATCgtGATT is a genetic change.
• Histone H4 K9gthistone H4 K9-CH3 is a potential
  epigenetic change because it might cause a
  heritable change by marking a site in the genome.
• Types of modifications
• Acetylation and methylation of lysine.
• Methylation of arginine
• Phosphorylation of serine
• Ubiquitination of lysine.
• The histone code hypothesis posits that serial
  modifications provide a blueprint for reading
  chromatin that can be used to regulated gene
  expression.

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Evidence for chromatin organization in the nucleus

• 1. Stoichiometric amounts of H2A, H2B, H3, and H4
  (H1 1/2). 2. X-ray
  analysis 10nm fibers.
  3. EM beads on a string.
  4. Micrococcal nuclease digestion
  periodic cleavage along the DNA. 5.
  Chemical cross-linking of (H3/H4)2.

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Micrococcal nuclease digestion of chromatin
Units Of nuclease
MW marker

• A ladder of DNA fragments differing by 200 bp

30 20 10
bp of DNA
800
600
400
200
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High resolution structure of a nucleosome core
particle
Note that the DNA tracks around the outer surface
of the nucleosome like the tread of a tire.
Arg/Lys
H3/H4
H2A/H2B

• Modeling of the DNA along the histone octamer
  surface of a nucleosome.

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Structure of a nucleosome core particle
containing DNA
Successive minor grooves have different widths.

• DNA contacts histones on only one side.

DNA
histones
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Histones are loaded onto the DNA by protein
chaperones
Cytoplasm nucleus
DNA synthesis
chaperone
nucleosome
Free histones octamer
transcription
H3.3, H2AZ etc variants
Nucleosomes containing variant histone H3, H2A
and histone modifications.
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Remodeling the nucleosomes
Changing the way nucleosomes bind to the DNA in
chromosomes is important to allow access to the
underlying DNA sequences during DNA replication,
repair, recombination, and transcription. This
occurs in three general ways Modification of
the lys and Arg residues on the histone tails
decreases the grip of the nucleosome on DNA
and causes the nucleosome to slide more
easily. Variant histones are added to
pre-existing nucleosomes ATP-dependent protein
remodelingcomplexes cause nucleosomes to
dissociate and/or slide along the DNA.
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Higher order structure of nucleosomes?
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Identifying and cloning functional units of
chromosome behaviour

• 1. Replication origins DNA sequences that allow
  initiation of replication
• 2. Centromeres DNA sequences that insure equal
  chromosome partition at mitosis and meiosis
  (point of attachment of kinetochores)
• 3. Telomeres DNA sequences at the ends of
  chromosomes that insure complete replication
• Identification of each of these elements required
  the construction of artificial chromosomes in
  bakers yeast-a model eukaryotic organism whose
  chromosomes have all of the hallmarks of those of
  larger eukaryotes but are 10-100X smaller.

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Artificial yeast chromosomes
Centromere (CEN 100 bp)
Circular
Gene for selection in yeast(HIS, TRP,
URA) (encoding an enzyme for synthesis of an
amino acid or pyrimidine)
Ori (yeast 60 bp)
Gene for selection in E. coli
(Amp)
poly-linker (unique restriction enzyme sites)
Ori (Ec)
Linear
Ori (yeast)
URA
CEN
Telomere (TEL)
Telomere (TEL 600 bp)
2000 - 20,000 bp
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Identifying and cloning functional parts of a
yeast chromosome
1. Cloning a yeast gene that complemented a
bacterial mutation a. yeast library in a
bacterial plasmid-----gtE. coli his-
mutant --------gtselect E. coli His
transformants-------gt b. characterize yeast DNA
insert it is homologous to E. coli HisG gene.
Therefore had cloned a yeast gene that functioned
in E. coli by complementation of the genetic
defect. 2. Transform a yeast His- mutant with
this plasmid. At a very low frequency His
transformants were found. The yeast cells had
taken up the plasmid and been genetically
transformed. ((Why the E. coli intermediate???-bec
ause the cloning step is inefficient and E. coli
is transformed more efficiently than yeast))
Where is the plasmid DNA in the cell? Two likely
possibilities 1. Replicating as a plasmid 2.
Integrated into a chromosome. To distinguish
these two possibilities isolate DNA from His
transformants and do a Southern blot
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Southern blot of yeast DNA
Treatment
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Sal1
Shows plasmid has Integrated into chromosome
HIS3 gene
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1.5
5
Sal1
Same size fragment because Sal1
digestion releases the HIS3 gene
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4.5
radioactive bands on the nitrocellulose membrane
Probe for Southern blot
Conclusion the plasmid had integrated into the
yeast chromosomal DNA by recombination with the
homologous but mutant his3 gene, implying that it
was unable to replicate on its own in yeast.
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Replication origins and centromeres
Other yeast genes were cloned in an analogous
manner, but some behaved differently because the
gene was tightly linked to an origin of
Replication, ori (known in yeast as an
ARS-Autonomous Replication Sequence). Cloned
centromeres conferred stability during mitotic
growth.
Yeast gene Location of Stability
during plasmid DNA mitotic
growth HIS3 chromosomal very
stable LEU2 chromosomal very
stable TRP1-ARS1 non- chromosomal
unstable TRP1-ARS1-CEN non-chromosomal very
stable
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Centromeres add mitotic stability to artificial
chromosomes
Centromeres enhance chromosome stability by
insuring that each daughter cell inherits one
and only one chromosome at each cell division
and also insure proper segregation at meiosis.
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Southern blot of yeast DNA when the plasmid
replicates as an episome
Yeast colonies
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Sal1
TRP1-ARS1 gene
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1.5
5
Sal1
2
4.5
Same size shows plasmid replicates autonomously
ie it hadnt integrated into a yeast chromosome
Probe for Southern blot
Conclusion the plasmid containing the TRP1-ARS1
region replicates autonomously (therefore it is
has an origin of replication associated with it).
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Replication origins, centromeres and telomeres
have unique DNA sequences

• Yeast origins of replication
• Yeast centromeres
• Telomeres Yeast (TG(1-3))n n200-300
• Human (T2AG)n n2000

(ATCAT)n
(ACCTA)n
(ATATAT)n
TG(1-3)n
TG(1-3)n
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ORIGINS OF REPLICATION, CENTROMERES AND TELOMERES
RECRUIT SPECIALIZED PROTEIN COMPLEXES
0.1-10X106 bp
105 bp
ORCORIGIN RECOGNITION COMPLEX (many/chromosome)
TELOMERASE COMPLEX (2/chromosome)
CENTROMERE BINDING COMPLEX (1/chromosome) )
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Summary

• The basic structural unit of chromosomes is the
  nucleosome
• A nucleosome contains an octamer of histones-a
  tetramer of (H3/H4), and two H2A/H2B dimers, and
  146 bp of DNA wound around the outside of the
  histones
• Chromatin remodeling occurs in multiple ways 1.
  covalent modification of the histones N-termini
  by enzyme complexes containing histone
  acetyltransferases and histone deacetylases, and
  2. ATP-dependent remodeling complexes that alter
  nucleosome positions/conformation on the DNA 3.
  Cohesins and condensins that condense and package
  chromosomes during the cell cycle.
• The functional units of the chromsomes-origins of
  DNA replication, centromeres, and
  telomeres-consist of specific and unique DNA
  sequences that bind multi-subunit protein
  complexes