DNA Replication

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material. Watson Crick 1953
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• Genetic information is stored in DNA
• The base-pairing principle is the basis of
  correctly transferring the information
• DNA (RNA) can be templates for replication.

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The Watson Crick Structure

• Two polynucleotide strands, right-handed twist to
  form a 20-Å-diameter double helix
• The helix has 10 base pairs per turn (34Å).

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The problems involved in the double helix model

• Double helix unwind?
• Semi-conservative?
• Replication starting point?
• Direction of replication?

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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The General Features(?) Double helix unwind
supercoil relaxing

• Topoisomerases
• Helicases
• Single strand DNA binding proteins (SSB)

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• Topoisomerase
• Induce negative super-coil at ori
• Relax positive super-coil
• Separate parental and daughter DNA strands.

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Type I topoisomerase creating transient single
strand breaks in DNA.
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Type II topoisomerase (gyrase) making transient
double strand breaks in DNA.
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• Helicases (Dna B protein)
• Helicases II III bind to lagging
  strand
• Rep protein binds to leading strand.

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• Single strand DNA binding protein (SSB)
• or DNA binding protein(DBP)
• Prevent hairpin structure formation
• Protect synthesed DNA from DNase digestion
• Stimulate DNA polymerases activity.

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The General Features(?) Semi-conservative
replication

• Proposed replication modelssemi-conservative
  replicationconservative replication
• The experiment by Meselson and Stahl (1957)

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The General Features(?) Origin of replication
Origin (ori) specific location(s) to control
replication start. Common features 1. origin
is a specific fragment 2. it contains repeat
sequences that can be recognised by some
proteins 3. AT rich.
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Replication folk (growing point) the point at
which replication is occurring.
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The General Features(?) Direction of replication

• Two origins, uni-directional replication
• One origin, uni-directional replication
• One origin, bi-directional replication

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The General Features(?) Semi-discontinuous
replication
The synthesis always proceeds in the 5' to 3'
direction.
How can both strands be synthesized
simultaneously?
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The General FeaturesSummary

• Double helix unwind supercoil relaxing
• Semi-conservative replication
• Origin of replication
• Direction of replication
• Semi-discontinuous replication

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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Prokaryotic DNA Replication

• Priming or initiation
• Elongation
• Termination

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Prokaryotic DNA Replication(?) Priming or
initiation
Double helix unwind supercoil relaxing

• Topoisomerases
• Helicases
• SSB

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Priming

• Primase (DnaG protein) catalyses formation of
  RNA primers for DNA synthesis.
• Preprimosome including several proteins (PriA,
  PriB, PriC, DnaB, DnaC and DnaT)

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• Primosome (primase preprimosome)
• binding to naked single strand DNA template
• moving along with replication forks on lagging
  strand
• being removed from the template and been reused
• DnaA protein E. Coli DNA replication

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Prokaryotic DNA Replication(?) Elongation

• A rapid reaction (500nt/sec in E. Coli).
• The reaction system template (DNA)
• substrates (dNTP) RNA primers and Mg
• enzymes (polymerases ligase)

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DNA polymerase I (pol I) isolated from E. Coli
by Kornberg in 1956

• Catalyses DNA synthesis (5' to 3') to fill the
  gaps (10nt/sec)
• 3' to 5' exonuclease excise mispaired nucleotide
  from the 3' end of the growing chain
• 5' to 3' exonuclease remove RNA primer, and edit
  mistakes by excising up to 10 nucleotides from 5'
  of a single strand.

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DNA polymerase II (pol II) discovered by
analysing a mutant E. Coli that has very little
pol I activity

• Catalyses DNA synthesis (5' to 3')
• 3' to 5' exonuclease to repair DNA damage.
• Lack 5' to 3' exonuclease activity

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DNA polymerase III (pol III) an important E.
Coli DNA replicase

• Polymerase activity (5' to 3' )
• 3' to 5' exonuclease activity but no nick.
• Lack 5' to 3' exonuclease activity
• Highly efficient gt150nt/sec
• Highly accurate error rate 1/106
• Highly continuous incorporate more than 5000 nt
  continuously.

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(d d' c y)2
A model for polymerase III
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The looping of the template for the lagging strand
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DNA ligase catalyses the formation of a
phosphodiester bond by attack of the 3-OH on the
a-phosphryl group, seal the nick and release AMP.
ATP ligase
ligase-AMP-P-5'-DNA
DNA-3'-O-P-O-5'-DNA AMP
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Prokaryotic DNA Replication(?) Termination

• The core sequence in termination region (ter)
  GTGTGGTGT
• Terminator utilization substance (Tus)
  contra-helicase function

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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Eukaryotic DNA Replication

• General features
• Replication and cell cycle
• Telomere and telomerase

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Eukaryotic DNA Replication(?) General features

• Genome size 108 - 109 base pair
• DNA protein complex
• Short primers and Okazaki fragments
• Multi-replication origin
• Bi-directional replication

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General features (continued)

• DNA polymerase activity low (incorporation
  rate 50nt/sec)
• More DNA polymerase molecules
• DNA polymerases ?, ?, ?, ?, and ?
• Polymerase activity (5' to 3' )
• 3' to 5' exonuclease activity

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General features (continued)

• Pol ? initiates replication with primase
• Pol ? and proliferating cell nuclear antigen
  (PCNA) complex is the major replicase
• Pol ? replicates the mitochondrial DNA
• Pol ? resembles pol ? and repairs damaged DNA
• Pol ? unknown its function.

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General features (continued)
Other factors involved in eukaryotic DNA
replication Replication factor A (RFA) or
replication protein A (RPA) acts as SSB to bind
a single strand DNA Replication factor C (RFC)
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Eukaryotic DNA Replication

• General features
• Replication and cell cycle
• Telomere and telomerase

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Eukaryotic DNA Replication(?) Replication and
cell cycle
DNA replication is critical to cell
proliferation DNA replication occurs at S phase
in eukaryotic organism cell cycle Phase
transition is controlled by some proteins and
factors.
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Cyclin
CDKs (cyclin dependent protein kinases)
protein phosphorylation (Rb protein)
Release transcript factor E2F
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Once per cell cycle
Cyclin B CDK1
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Eukaryotic DNA Replication(?) Telomere and
telomerase

• Telomere
• Specialized ends on eukaryotic chromosomes
• Consist of repetitive oligomeric sequences
• Gy(T/A)x y gt1, x 1-4
• Human telomere GGGTTA

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Telomerase A modified reverse transcriptase at
telomere A ribonucleoprotein complex Elongate
the lagging strand template from its 3?-hydroxyl
end and complete replication of chromosomal
telomere.
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• Biological significance of telomere and
  telomerase
• Completion of replication for chromosomal
  telomere
• Protection for the end of DNA strands
• Stabilizing chromosomes to prevent aging.

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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Reverse Transcription

• The observation of reverse transcription
• The discovery of reverse transcriptase

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• The discovery of RSV (Rous Sarcoma
  Virus)(Published on J. Exp. Med. in 1911) RSV
  is RNA virus
• Temins observation the blockage of RSV
  propagation by DNA synthesis inhibitors
• Proposed provirus (1963).

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Peyton Rous 1966 Nobel prize winner for his
discovery of tumour inducing viruses
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The isolation of reverse transcriptase, 1970 (by
Temin and Baltimore, independently)

• RNA template RNA-DNA hybrid
• DNA template
• DNA-DNA hybrid

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• 5' 3' DNA synthesis
• Primer tRNA
• Present of Zn
• Lack of 3' 5' exonuclease activity

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DNA Replication

• The general features
• Prokaryotic DNA replication
• Eukaryotic DNA replication
• Reverse transcription
• DNA damage and repair

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DNA Damage and Repair

• Proofreading by DNA polymerase
• Environmental base damage and repair
• Mechanism of damage repair
• Damage repair for eukaryotic DNA.

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Proofreading by DNA Polymerases

• Errors occur during DNA replication
• The replication stops
• DNA pol I and pol III (pol d) excise mismatched
  bases (3' 5' exonuclease activity).

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Environmental Base Damage Repair
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Mechanism of Damage Repair

• Excision repair AP endonuclease UvrABC UvrA
  and UvrB binding to damaged DNA UvrC excise
  damaged bases Helicase unwind the double strand
• DNA synthesis (pol I) and DNA ligation

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• Recombination repair
• SOS repair

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Damage repair for Eukaryotic DNA

• Xeroderma pigmentosum (XP)
• First described by Hebra and Kaposi
• Autosomal recessive inherited disease
• Defect in the excinuclease that hydrolyzes the
  DNA backbone near a pyrimidine dimer
• Highly sensitive to UV induced damage.

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Damage repair for Eukaryotic DNA

• DNA repair genesradiation sensitive (RAD) gene
• Rad 3 encode helicase.
• Rad 10 encode proteins similar to UvrA and UvrC.