DNA Replication
Overview
Mechanism in Bacteria
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Semiconservative Replication
• First: RNA molecules that could self-replicate
• Later: DNA
– DNA Replication more complex
– Required a large number of auxiliary components
– DNA is template, but not enzyme
• Watson & Crick (1953) - proposed DNA structure
– Replication by strand separation
– Both strands have same information
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Figure 13.1
Semiconservative Replication
• Watson & Crick predicted
• Other Possibilities
– Conservative - 2 original strands stay together
– Dispersive - parental strand integrity disrupted
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Figure 13.2
Semiconservative Replication
• Matthew Meselson & Franklin Stahl (1958,
Caltech)
– Grow bacteria in media with 15NH4Cl
– Bases contain "heavy" nitrogen
– Wash out 15NH4Cl; put bacteria in 14NH4Cl ("light")
– Extract DNA & do CsCl density-gradient
– one generation: all DNA is hybrid density
– two generations, half DNA is light & half is hybrid
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Figure 13.3a
Semiconservative Replication
• Eukaryotes also semiconservative
– BrdU replaces thymidine in DNA
– one generation: all chromosomes hybrid with
label
– two generations, half chromosomes are double
labeled & half hybrid
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Figure 13.4a
Genetic & Biochemical Dissection
• Availability of mutants
– how can such cells be cultured?
– ts mutants
• in vitro replication systems
– Replicate DNA with extracts
– Remove specific proteins (antibodies) or
– Add purified proteins whose activity is to be tested
• at least 30 proteins needed for E. coli
replication
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Bidirectional Replication
• Replication origins
– oriC ; ~245 bp sequence on bacterial chromosome
– Proteins (a number) bind at oriC to initiate replication
– Replication is bidirectional
• Replication forks
– Parental helix is undergoing strand separation
– Complementary strand synthesis
– Forks meet across from origin, where replication is
terminated
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Figure 13.5
The Topology of Unwinding
• Analogy: two-stranded helical rope
– Circular & linear DNAs not free to rotate
– Tension not relieved
– DNA can become tightly overwound
– Positive Supercoiling (ahead of fork)
– E. coli : ~400,000 turns replicated by 2 forks in
~40 min
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Figure 13.6
The Topology of Unwinding
• Topoisomerases relieves mechanical strain
– DNA gyrase can relieve stress
– Generates Negative Supercoils
– Mechanism
• Cleaves both duplex strands
• Passes DNA through double-stranded break
• Seals cuts; driven by ATP hydrolysis
– Similar enzymes are found in eukaryotes
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DNA Polymerase Properties
• DNA polymerase I (E.coli)
– incorporates labeled DNA precursors into
polymer
– Requires template, dTTP, dATP, dCTP, dGTP)
– New DNA had same composition as original
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DNA Polymerase Properties
• DNA polymerase reaction similar to RNA
polymerase, but...
– DNA not RNA, requires primer
– Single-stranded DNA alone does not work
– All DNA polymerases require primer & template
– Polymerizes DNA in 5' to 3' (5'—>3') direction
– How is synthesis initiated in cell without DNA
primer?
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Figure 13.7
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Figure 13.8
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Figure 13.8
DNA Polymerase Properties
• DNA polymerases (II & III) were
discovered with mutants of Pol I
– Pol I mutant had <1% of normal pol activity
– but multiplied at normal rate!
– Typical bacterium
• ~300 - 400 copies of DNA Polymerase I
• ~40 copies of DNA Pol II
• 10 copies of DNA Pol III
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Semi-discontinuous Replication
• both strands at fork synthesized 5’ to 3’
– Leading strand (toward fork) made
continuously
– Lagging strand (away from fork) made in
fragments
– Okazaki fragments must wait for parental
strands to separate
– Later, these fragments are linked together
– Both strands probably made simultaneously
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Figure 13.9
Semi-discontinuous Replication
• Reiji Okazaki (Nagoya Univ.; Japan)
– brief pulses of 3H-thymidine
– Label in DNA fragments (~1000 - 2000
nucleotides long)
– pulse then chase: label in much larger DNA
– Joined by DNA ligase
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Figure 13.10
Semi-discontinuous Replication
• But DNA polymerase requires primer
– Enzyme makes short RNA primer for initiation
– Leading strand initiated at origin by an RNA
polymerase
– Okazaki fragments also initiated by primase
– Short RNA primers eventually removed
– gap is filled with DNA & sealed by DNA ligase
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Figure 13.11
Solving the Topological Problem
• DNA gyrase
– Negatively supercoils DNA
– Genome and Plasmids maintained in Negatively
Supercoiled state
– DNA already “primed” to unwind & separate
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Solving the Topological Problem
•
Helicase (E. coli has at least 12)
– DnaB helicase, unwinding machine during replication
– Fired by ATP hydrolysis
– Break H bonds holding 2 strands together
– 6 subunits arranged to form ring-shaped protein
– Encircles a single DNA strand
– First attaches to DNA at replication origin
– Helped by DnaC
– moves along lagging-strand template unwinding helix
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Solving the Topological Problem
• SSB proteins (single strand binding
proteins)
– SSBs bind selectively to single-stranded DNA
– Keep it extended & prevent it from being
rewound
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Figure 13.12a
Primosome = primase + helicase
• Helicase moves processively
– primase periodically binds to helicase
– initiates synthesis of short RNA primers
– Primers extended as DNA by a DNA polymerase
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Primosome = primase + helicase
• Does same DNA polymerase III synthesize
Okazaki fragments?
– Replicate DNA in vitro and suddenly dilute reaction
– Reaction does not slow
– Hitches a ride with DNA polymerase going other way
– 2 polymerases are part of a single protein complex
– DNA of the lagging strand loops back on itself
– When Okazaki fragment finished, lagging-strand
released
– trombone model: looping DNA grows & shortens
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Figure 13.13
Roles of DNA Polymerases
• DNA polymerase I
– mostly for DNA repair to correct damaged DNA
– single subunit
– removes RNA primers at 5' Okazaki fragment end
– replaces the RNA with DNA
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Roles of DNA Polymerases
• DNA polymerase II
– Role uncertain
– mutants have no evident deficiency
• DNA polymerase III (replicase)
– acts in DNA replication in E. coli
– part of large complex: DNA polymerase III
holoenzyme
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DNA Polymerase III Structure
• holoenzyme much larger than other two
polymerases
• 10 different subunits
• b subunit “clamp” and multisubunit
“clamp loader”
• Clamp loader required to assemble b
clamp
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Figure 13.14
DNA Polymerase III Structure
• b subunit – a noncatalytic subunit
– keeps the polymerase associated with the DNA
template
– stong attachment required for processivity
– loose attachment required to move along template
– conflicting properties: balance
– b subunits (2) form a doughnut-shaped complex
around DNA
– sliding clamp allows processive movement
– Pol III cycled to new b clamp waiting at RNA primer
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Figure 13.15b
DNA Polymerases and
Exonucleases
• Exonucleases
• enzymes that degrade nucleic acids
• remove 1 or more terminal nucleotides at a time
• Kornberg first to observe in DNA Polymerases
• Due to contaminating enzyme? No.
• Exonucleases can be 5'—>3' or 3'—>5'
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Figure 13.16
DNA Polymerases and
Exonucleases
• DNA pol I has both 5'—>3' & 3'—>5' exo
– 3 different domains of enzyme
– The 2 exonuclease activities have entirely
different roles in replication
• Pol I 5'—>3' exonuclease activity
degrades both RNA & DNA
– removes RNA primer at Okazaki fragment 5' end
– polymerase fills in the resulting gap
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DNA Polymerases and
Exonucleases
• Pol I 3'—>5' exonuclease does proofreading
– Error rate ~10-9 (~1 out of 1 billion nucleotides)
– < 1 nucleotide alteration/every 100 replication cycles
– Since E. coli genome has ~4 x 106 nucleotide pairs
– Wrong base once every 105 - 106 nucleotides
– Exo removes most of these (Pol stalls)
– Raises fidelity to 10-7 - 10-8
– This activity is found in DNA polymerase I, II, & III
– Mismatch repair which takes error rate to ~10-9
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Figure 13.17
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Figure 13.18a
DNA Replication Fidelity
• Fidelity of DNA replication can thus be
traced to 3 distinct activities
– Accurate selection of nucleotides: 10-6
– Immediate proofreading: 10-2
– Post-replicative mismatch repair: 10-1
– Total spontaneous mutation rate: 10-9
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