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Requirements for the Genetic Material
• 1. Replication
Reproduced and transmitted faithfully from cell to cell-generation
to generation.
• 2. Information Storage
Biologically useful information in a stable form.
• 3. Expression of Information
Express itself: Other biologically important molecules, and
ultimately cells and organisms, will be produced and maintained.
• 4. Variation (by mutation)
Capable of variation: some change is required
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REPLICATION
OF DNA
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Replication of DNA
• What is the mode of DNA
replication and how was
that determined?
Fig. 10-1
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Replication of DNA: 3 possible models
Fig. 10-2
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Meselson-Stahl Experiment
• Grew E. coli in medium containing ammonium chloride (15NH4Cl) as sole N source – 15N = “heavy” but nonradioactive isotope
– After multiple generations, all N-bases of DNA contain 15N
– Extracted DNA and centrifuged in density gradient (CsCl)
• Grew 15N labelled cells in regular medium (14NH4Cl) – Collected samples after 1st, 2nd and 3rd generations
– Extracted DNA and centrifuged in density gradient (CsCl)
• Compared banding patterns observed in density gradients
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Meselson-Stahl Experiment
Fig. 10-3
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In the Meselson-Stahl experiment, if DNA is
replicated conservatively, then after 2
generations of replication there would be…
• A) one “old” band and one “new” band
• B) one “hybrid” band
• C) one “hybrid” band and one “new” band
• D) one “hybrid” band and one “old” band
• E) one “old” band, one “hybrid” band, and one “new” band
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DNA Replication • Bacterial & eukaryotic models
– Similar process:
• Initiation
• Elongation
• Termination
– Differences due to differences in DNA
structure and complexity of DNA coiling
• Circular vs. linear DNA
• Single vs. multiple origins of replication
• Absence or presence of nucleosomes
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DNA Replication in Bacteria
Fig. 10-6
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DNA Replication
• Unwind & denature double helix – Helicases
• Unwind, open & stabilize helix
• DnaA, DnaB, DnaC
– Stabilized by SSB’s (single-stranded binding proteins)
– Problem • Unwinding creates
supercoiling causes torsional stress
• Relieved by DNA gyrase
• SS or DS nicks
Fig. 10-9
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DNA Replication
• Initiate synthesis
– RNA primase (RNA polymerase) adds an
RNA primer
• ~5-15 nucleotides
Fig. 10-9
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The role of the DnaA protein in bacterial DNA
replication is to…
• A) prevent renaturation of the two DNA strands during replication
• B) synthesize and RNA primer
• C) relieve the tension of supercoiling
• D) initially unwind the DNA double helix
• E) detect replication errors
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DNA Replication
• Chain elongation
– DNA pol III
• Requires free 3’-OH to bind
• Adds nucleotides 5’ 3’
Fig. 10-9
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Fig. 10-7
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Elongation
• Elongation is...
– Simultaneous
• Both strands at once
– Bidirectional
• Strands are antiparallel
– Continuous & discontinuous
processes
• Leading strand (continuous)
• Lagging strand (discontinuous
– Okazaki fragments
Fig. 10-11
simultaneous synthesis
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Okazaki fragments are a consequence of…
• A) the inability of DNA polymerase to initiate a new DNA strand
• B) the inability of DNA polymerase to correct replication errors
• C) random strand breakage due to supercoiling
• D) mutations in the gene for DNA ligase
• E) the inability of the DNA polymerase to synthesize in the 3’-5’ direction
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DNA Replication
• Primers removed and lagging strand gaps
filled
– DNA pol I
• Cleaves out RNA primer and replaces with DNA
• Has both 5’ 3’ exonuclease and polymerase
activities
– DNA ligase
• Forms final phosphodiester bond to fill gap
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Chain Elongation
• Enabling of concurrent synthesis
– DNA pol III is a dimer
– Loop forms to keep antiparallel template
strands effectively 3’ 5’ relative to DNA pol III
• 1000-2000 bp
Fig. 10-12
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Proofreading
• Critical that new strand is exact
complement of template
• DNA pol I & III have 3’ 5’ exonuclease
activity
– Recognition & replacement of mismatches
during elongation
– “back-up” and replace mismatched bases
• DNA pol II
– Active in DNA repair to external damage
(i.e., UV light)
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Polymerase Comparison
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Bacterial DNA Replication
Summary
Fig. 10-13 dna replication
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All known bacterial DNA polymerases…
• A) can initiate DNA chain synthesis
• B) have 5’ to 3’ polymerization activity
• C) have 5’ to 3’ exonuclease activity
• D) have 3’ to 5’ polymerization activity
• E) all of the above
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Replication of Eukaryotic DNA
• Problems
– Larger genomes
– Eukaryotic DNA pols are slower
– Nucleosomes
– Linear chromosomes
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Replication of Eukaryotic DNA
• Solutions
– Multiple origins of replication
• Yeast : 250-400 replicons
• Mammals: up to 25,000 replicons
– More types (14) of polymerases
• Different activities / operate under different conditions
– More polymerase molecules per cell
• E. coli ~400/cell
• Homo sapiens ~50,000/cell
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Comparison of Replication
Rates
• E. coli 4.7 kb ~20-40 min
• Drosophila 120 kb ~ 3 min
• Homo sapiens 3300 kb ~ 7 hrs
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Replication of Eukaryotic DNA
• Replication problems at
ends (telomeres) of linear
chromosomes
– RNA primer at terminal end
• Once removed, no 3’-OH
available for addition of DNA
nucleotides
• Can lead to telomere
shortening (cellular clock)
– Fetal tissue culture cells - 60-
80 divisions max.
– Adult cells - 10-20 divisions
max Fig. 10-16
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Telomeres
• Repeated sequences
– Tetrahymena – a protozoan
• TTGGGG tandem repeats
• Overhang on G-rich strand of 12-16 bases
• G-quartets – form loops on ends of chromosomes
Fig. 10-17
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Telomerase
• A ribonucleoprotein – Contains RNA (-AACCCCAAC-)
• Recognizes telomeric sequence and adds repeats – RNA primase
– DNA pol I & ligase
• Typically low activity in somatic cells; high activity in cancerous cells
Fig. 10-17
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Telomerase…
• A) is a ribonucleoprotein
• B) has reverse transcriptase activity
• C) adds short tandem repeats to the ends of chromosomes during DNA replication
• D) functions to replicate DNA at the ends of linear chromosomes
• E) all of the above
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Recombination (Crossing Over)
1. Homologs pair and synapses form
2. Endonucleases “nick” DNA at adjacent sites on both homologs – SS
3. Ends are displaced and pair with homologous sequence on other duplex
Fig. 10-18
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Recombination
4. Ligase seals nicks
– Creates heteroduplex
5. Branches migrate
– H-bonds “unzip” and form
complementary bonds
with other duplex
6. Duplexes separate
& rotate
– Chiasmata
Fig. 10-18
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Recombination
7. Endonucleases nick
opposite sides of
chi form
8. Homologs are ligated
and separated
Fig. 10-18
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Recombination…
• A) is a ribonucleoprotein
• B) has reverse transcriptase activity
• C) adds short tandem repeats to the ends of chromosomes during DNA replication
• D) functions to replicate DNA at the ends of linear chromosomes
• E) all of the above
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Gene Conversion
• Mismatch after crossover can result in
mutation
Fig. 10-19