DNA replication

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DNA replication

• Chapter 16

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Figure 16.1
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History of DNA

• Griffith
• Mice Strep
• Transformation
• External DNA taken in by cell

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Experiment
Living R cells(nonpathogeniccontrol)
Mixture of heat-killed S cells andliving R cells
Living S cells(pathogeniccontrol)
Heat-killed S cells(nonpathogeniccontrol)
Results
Mouse dies
Mouse healthy
Mouse dies
Mouse healthy
Living S cells
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History of DNA

• Hershey-Chase
• Bacteriophages
• Supported heredity information was DNA

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Bacteriophages
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Figure 16.4
Experiment
Batch 1 Radioactive sulfur (35S) in phage protein
Labeled phagesinfect cells.
Agitation frees outsidephage parts from cells.
Centrifuged cellsform a pellet.
Radioactivity(phage protein)found in liquid
Radioactiveprotein
Centrifuge
Pellet
Batch 2 Radioactive phosphorus (32P) in phage DNA
RadioactiveDNA
Centrifuge
Radioactivity (phageDNA) found in pellet
Pellet
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History of DNA

• Franklin
• X-ray diffraction
• Double helix
• Watson-Crick
• Double helix model

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History of DNA Duplication

• Meselson and Stahl
• Bacteria
• 14N and 15N
• Semiconservative method.

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Fig. 16-10
First replication
Second replication
Parent cell
(a) Conservative model
(b) Semiconserva- tive model
(c) Dispersive model
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Figure 16.11
Experiment
Bacteria cultured in medium with 15N(heavy
isotope)
Bacteria transferred to medium with 14N(lighter
isotope)
Results
DNA samplecentrifugedafter firstreplication
DNA samplecentrifugedafter secondreplication
Less dense
More dense
Conclusion
Predictions
First replication
Second replication
Conservativemodel
Semiconservativemodel
Dispersivemodel
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DNA structure
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Nucleic acid structure

• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• Nucleotides

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Nucleotide structure

• 1. 5 carbon sugar (ribose)
• 2. Phosphate
• 3. Nitrogenous base

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Nucleotide structure
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Nitrogenous base

• Purines (2 rings)
• Adenine(A) Guanine(G)
• Pyrimidines (1 ring)
• Cytosine (C), Thymine (T) DNA only
• Uracil (U) RNA only

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Phosphodiester bondLinks 2 sugars (nucleotides)
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Nucleic acids

• 5 Phosphate group (5C) at one end
• 3 Hydroxyl group (3C) at the other end
• Sequence of bases is expressed in the 5 to 3
  direction
• GTCCAT 5pGpTpCpCpApT---OH 3

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Double helix

• Complementary
• Sequence on one chain of DNA
• Determines sequence of other chain
• 5-ATTGCAT-3
• 3-TAACGTA-5

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Double Helix

• Complementary
• Purines pair with pyrimidines
• Diameter of base pairs are the same
• Adenine (A) forms 2 hydrogen bonds with Thymine
  (T)
• Guanine (G) forms 3 hydrogen bonds with cytosine
  (C)

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Double Helix

• Sugar-phosphates are the backbone
• Complementary
• Phosphodiester bonds
• Strands are antiparrellel
• Bases extend into interior of helix
• Base-pairs form to join the two strands

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Fig. 16-7
5? end
Hydrogen bond
3? end
1 nm
3.4 nm
3? end
0.34 nm
5? end
(b) Partial chemical structure
(a) Key features of DNA structure
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Duplication

• DNA unzips-breaks hydrogen bonds
• New strand forms based on existing strand
• Old strand is saved
• Compliment of new strand
• New DNA-one old strand one new strand
• Semiconservative replication

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Fig. 16-9-3
A
A
T
T
A
T
T
A
C
C
G
G
G
C
G
C
A
T
A
A
T
A
T
T
T
T
A
T
T
A
A
A
C
C
G
C
C
G
G
G
(c) Daughter DNA molecules, each consisting of
one parental strand and one new strand
(b) Separation of strands
(a) Parent molecule
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Duplication Enzymes

• DNA helicase
• Enzyme opens helix starts duplication
• Separates parental strands
• Single-strand binding protein
• Binds to unpaired DNA
• After separation
• Stabilizes DNA

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Duplication Enzymes

• DNA polymerases
• Help lengthen new strand of DNA
• Adds new nucleotides strand
• Synthesis occurs only one direction
• 5 to 3
• Adding new nucleotides to the 3OH

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Duplication Enzymes

• Primer
• Section of RNA
• Complementary to the parental DNA
• Synthesis occurs only one direction
• 5 to 3
• DNA primase
• Enzyme creates the primer

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Duplication Enzymes

• Topoisomerase
• Relieves strain of unwinding DNA
• DNA pol1
• Removes primers
• Replaces with DNA nucleotides
• DNA ligase
• Creates phosphodiester bonds between Okazaki
  fragments

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Table 16.1
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Duplication

• OriC
• Origins of replication
• Starting point in DNA synthesis
• Replication is bidirectional
• Proceeds in both directions from origin
• 5to 3direction

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Duplication

• E coli (bacteria)
• Circular DNA
• One origin
• Eurkaryotes
• Multiple origins

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Origins of Replication
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Duplication

• Replication bubble
• Separation of strands of DNA
• Replication of DNA
• Replication fork
• Y-shaped region
• End of replication bubble
• Site of active replication

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Duplication
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Duplication
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DNA Replication Overview
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Duplication

• Leading strand
• DNA continuous 5 to 3 replication (towards
  fork)
• Template is 3 to 5
• Lagging strand
• DNA duplicated in short segments (away from fork)
• Okazaki fragments
• Short stretches of new DNA-lagging side

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Duplication

• Unzips (helicase, single-strand binding protein,
  topoisomerase)
• Primer
• DNA polymerase (5to3)
• DNA ligase

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Duplication
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Fig. 16-14
New strand 5? end
Template strand 3? end
5? end
3? end
Sugar
T
A
A
T
Base
Phosphate
C
G
G
C
G
G
C
C
DNA polymerase
3? end
A
A
T
T
3? end
C
C
Pyrophosphate
Nucleoside triphosphate
5? end
5? end
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Leading Strand
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Fig. 16-13
Primase
Single-strand binding proteins
3?
Topoisomerase
5?
3?
RNA primer
5?
5?
3?
Helicase
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Fig. 16-15b
Origin of replication
3?
5?
RNA primer
5?
Sliding clamp
3?
5?
DNA pol III
Parental DNA
3?
5?
5?
3?
5?
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Fig. 16-16a
Overview
Origin of replication
Leading strand
Lagging strand
Lagging strand
2
1
Leading strand
Overall directions of replication
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Lagging Strand
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Fig. 16-17
Overview
Origin of replication
Lagging strand
Leading strand
Leading strand
Lagging strand
Single-strand binding protein
Overall directions of replication
Helicase
Leading strand
DNA pol III
5?
3?
3?
Primer
Primase
5?
Parental DNA
3?
Lagging strand
DNA pol III
5?
DNA pol I
DNA ligase
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3?
5?
3
1
2
3?
5?
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Fig. 16-16
Overview
Origin of replication
Lagging strand
Leading strand
Lagging strand
2
1
Leading strand
Overall directions of replication
5?
3?
3?
5?
Template strand
RNA primer
3?
5?
3?
1
5?
3?
Okazaki fragment
5?
3?
1
5?
5?
3?
3?
2
5?
1
5?
3?
3?
5?
1
2
5?
3?
3?
5?
1
2
Overall direction of replication
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Figure 16.18
Leading strand template
DNA pol III
Leading strand
Parental DNA
5'
3'
3'
5'
3'
3'
5'
5'
Connecting protein
Helicase
DNA pol III
3'
5'
Lagging strandtemplate
Lagging strand
3'
5'
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Duplication
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Duplication
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Duplication

• Telomers
• Sequences at ends of chromosomes
• Short nucleotide sequences
• Repeated 100-1000 times
• Prevents 5 end erosion
• Telomerase
• Enzyme that lengthens telomers
• Usually in germ cells

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Repairs

• Mismatched pair
• Duplication error
• Enzymes remove error
• Nucleotide excision repair
• Damaged section removed
• Nuclease
• New nucleotides fill gap
• Complement DNA section not damaged

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Figure 16.19-3
5'
3'
3'
5'
Nuclease
5'
3'
3'
5'
DNApolymerase
5'
3'
5'
3'
DNAligase
3'
5'
3'
5'
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Chromosome packaging

• Chromatin
• Complex composed of DNA and proteins
• 40 DNA 60 protein
• Heterochromatin
• More compacted chromatin
• Euchromatin
• Loosely packed chromatin

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Figure 16.23
5 µm
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Chromosome packaging

• Double helix
• Histones proteins
• Nucleosome DNA coiled around 8 histones (10nm)
• Nucleosomes then coil (30nm)
• Looped domains attach to chromosome scaffold
  (300nm)
• Domains coil form chromosome

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Figure 16.22
Chromatid(700 nm)
Nucleosome(10 nm in diameter)
DNAdouble helix(2 nm in diameter)
30-nm fiber
Scaffold
Loops
H1
Histone tail
300-nmfiber
Histones
DNA, thedouble helix
Nucleosomes,or beads ona string(10-nm fiber)
Histones
30-nm fiber
Replicatedchromosome(1,400 nm)
Loopeddomains(300-nm fiber)
Metaphasechromosome
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Figure 16.22a
Nucleosome(10 nm in diameter)
DNAdouble helix(2 nm in diameter)
H1
Histone tail
Histones
DNA, thedouble helix
Histones
Nucleosomes, or beads ona string (10-nm fiber)
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Figure 16.22b
Chromatid(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
30-nm fiber
Replicatedchromosome(1,400 nm)
Looped domains(300-nm fiber)
Metaphasechromosome
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DNA Packing