UNIT 11 DNA REPLICATION

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UNIT 11DNA REPLICATION
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SEMICONSERVATIVE REPLICATION
MESSELSON-STAHL EXPERIMENT
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http//en.wikipedia.org/wiki/Meselson-Stahl_experi
ment
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The Meselson-Stahl experiment, which showed that
DNA replicates semiconservatively
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Chinese hamster cells after two rounds of
replication in 5-Bromodeoxyuridine followed by
staining (BubR stains less intensely)
2 chromatids
2 chromatids 2nd round of division
After division is completed, 1 chromosome
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REPLICATION INITIATION IN PROKARYOTES
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Prokaryote DNAs have a single origin of
replication
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DnaA boxes are 9 13bp sequences found at the
origin of replication ori and are AT-rich
DNA B protein
Helicases are enzymes that disrupt the hydrogen
bonds between DNA strands.
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REPLICATION INITIATION IN EUKARYOTES
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Yeast cells
OriC (Origin of Replication C) recognizes the
100-200bp AT-rich origin of replication and binds
to adjacent site
OriC recruits two additional proteins, then
helicase
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Interestingly, in higher eukaryotes, origins of
replication can be very long, (tens to hundreds
of thousands of nucleotides) and are genetically
divergent, perhaps allowing differential timing
of replication of chromosome areas
ORC
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Replicating DNA of Drosophila melanogaster
EUKARYOTIC CHROMOSOMES HAVE MULTIPLE ORIGINS OF
REPLICATION
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SEMICONSERVATIVE REPLICATION
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DNA chain elongation catalyzed by DNA polymerase
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(Primase is an RNA polymerase)
(8-12 nucleotides)
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3
5
3
5
opening replication fork
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DNA ligase seals the gap
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Topoisomerases manage supercoiling by breaking
one or more of the DNA backbones
Eukaryote
Prokaryote
DNA gyrase is a topoisomerase
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PolIII holozyme PolIII plus clamp and other
proteins
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NUCLEOSOMES (Eukaryotic replication)
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ASSEMBLY OF NEW DNA INTO NUCLEOSOMES (Eukaryotes
only)
When eukaryotic DNA is replicated, it complexes
with nucleosomes. This requires synthesis of
histone proteins and assembly of new nucleosomes.
Transcription of histone genes is initiated
near the end of GI phase, and translation of
histone proteins occurs throughout S phase.
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ASSEMBLY OF NEW DNA INTO NUCLEOSOMES (Eukaryotes
only)
Newly replicated DNA is organized into
nucleosomes very quickly. DNA synthesis requires
nucleosomes to disassemble so the DNA can be
denatured, but only 200-300 bp will be
nucleosome-free around the replication forks.
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ASSEMBLY OF NEW DNA INTO NUCLEOSOMES (Eukaryotes
only)
Data strongly suggest that new nucleosomes are
composed of entirely new histones, and existing
nucleosomes are conserved. Old and new
nucleosomes bind randomly to the sibling
chromatids after replication.
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Chromatin assembly factor 1 (CAF-1) delivers new
histone proteins to the replication fork to
assemble new nucleosomes in prokaryotes. Old
nucleosomes are split between the strands.
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THE TELOMERE PROBLEM IN EUKARYOTES
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The problem of replicating completely a linear
chromosome in eukaryotes
An RNA primer is synthesized at the 5 end of the
chromosome
Removal of the primer leaves a gap that cant be
filled by DNA polymerase
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a
b
c
d
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e
f
g
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Telomeres of eukaryotes have caps
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The link between telomere length and aging

• Somatic cells have little telomerase and as cell
  division occurs, chromosomes get shorter. (Germ
  cells have ample telomerase)
• People with Werner syndrome age prematurely
• Short telomeres caused my mutation in telomerase
  cap protein Mutation in wrn
• Dyskeratosis
• Mutations in genes for telomerase activity

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Werner syndrome at ages 15 and 48
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REPLICATION OF VIRAL DNA
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The replication process of double-stranded
circular DNA molecules through the rolling circle
mechanism
New strand 5-gt3
New strand 3-gt5 strand filled in
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• Retroviruses Some RNA viruses use a specialized
  enzyme, reverse transcriptase, to copy dsRNA into
  a DNA strand which acts as a template for
  replication and can integrate into a eukaryotic
  chromosome.
• HIV is a retrovirus

When retroviruses have integrated their genome
into the germ line, their genome is passed on to
a following generation. These endogenous
retroviruses, contrasted with exogenous ones, now
make up 8 of the human genome.
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PRACTICE
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The conservative model of replication is when

• an intact double helix is used as a template for
  a new double helix
• Each helix of a double helix forms a new double
  helix
• DNA replication occurs in small pieces

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In a cesium chloride gradient, DNA labeled with
14N would appear at the ____of the gradient
after centrifugation

• Top
• Middle
• Bottom

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Helicases

• Unwind supercoiling
• Disrupt bonds at ori sequences
• Keep helices separated
• Ligate sugar phosphate backbones

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In eukaryotes, DNA replicates at

• several places along a chromosome
• at one place on each chromosome

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DNA polymerase synthesizes new DNA

• 5 to 3 only
• 3 to 5 only
• in both directions

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Okazaki fragments are

• fragments from DNA replication on one of two
  helices
• Fragments from DNA replication on both helices
• Fragments from the ends of chromosomes

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Topoisomerases

• Unwind supercoiling
• Disrupt bonds at ori sequences
• Keep helices separated
• Ligate sugar phosphate backbones

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Nucleosomes

• Are distributed randomly to newly synthesized DNA
• Are composed of a mix of old histone proteins and
  new histone proteins
• Are completely removed from the chromosome before
  replication

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Without telomerase -

• chromosomes could not attach to the spindle
• chromosomes could not replicate
• chromosomes would get shorter

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END
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Synthesis of telomeric DNA by telomerase
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• Five DNA polymerases are known in mammalian
  cells
• a (alpha) is nuclear, uses RNA primers, is
  involved in nuclear DNA replication and has not
  been shown to proofread.
• ß (beta) is nuclear, serves in DNA repair and
  does not show proof- reading activity.
• d (delta) is nuclear, uses RNA primers, is
  involved in nuclear DNA replication and is
  capable of proofreading.
• ? (gamma) is mitochondrial, replicating the
  mitochondrial DNA using RNA primers and
  proofreading.
• e (epsilon) is nuclear, has proofreading
  activity and may be used for DNA repair