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Chapter 13
DNA Replication
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13.1 Introduction
topoisomerase An enzyme that changes the numberof times the two strands in a closed DNA molecule cross
each other.
It does this by cuttingthe DNA,passingDNA throughthe break, andresealingthe DNA.
replisome The multiprotein structure that assembles atreplication forks to undertake synthesis of DNA.
It contains DNA polymeraseand other enzymes.
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13.2 DNA Polymerases Are the Enzymes That Make DNA
A bacterium or eukaryotic cell has several different DNApolymerase enzymes.
! However, they share same activity (i.e., DNA synthesis)! Synthesis from 5 to 3 from a template that is 3 to 5.
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13.2 DNA Polymerases Are the Enzymes That Make DNA
Figure 13.03
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13.2 DNA Polymerases Are the Enzymes That Make DNA
A bacterium or eukaryotic cell has several different DNApolymerase enzymes.
! Some are responsible for de novo synthesis of new DNAstrands.
! Other are involved in the repair of damaged DNA (removal ofshort stretch of damaged region and synthesis of new DNA).
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13.2 DNA Polymerases Are the Enzymes That Make DNA
Figure 13.04
Figure 13.21
E. coliEukaryotes
Replicases: high-fidelity
Error-prone polymerases
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13.4 DNA Polymerases Control the Fidelity of
Replication
DNA replication error in bacteria: 10-8to 10-10(equivalentto ~1 error per 1000 replications).
Proofreading a mechanism for correcting errors inDNA synthesis"wrong nucleotide is removed by 3-5exonuclease activity of DNA polymerases and a correct
nucleotide is added.
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13.4 DNA Polymerases Control the Fidelity of
Replication
Figure 13.05
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13.4 DNA Polymerases Control the Fidelity of
Replication
Processivity the ability of an enzyme to performmultiple catalytic cycles with a single template instead of
dissociating after each cycle.
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13.6 The Two New DNA Strands Have Different
Modes of Synthesis
The DNA polymerase advances continuously when it synthesizesthe leading strand (5!3!), but synthesizes the lagging strand by
making short fragments (Okazaki fragments) that are subsequently
joined together.
Figure 13.08
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13.7 Replication Requires a Helicase and Single-
Strand Binding Protein
Replication requires a helicasetoprocessivelyseparatethe strands of DNA using energy provided by hydrolysisof ATP.
A single-stranded binding protein (SSB) cooperativelybinds to single stranded DNA, which is required tomaintain the separated strands.
Figure 13.09: A hexamerichelicase moves along one
strand of DNA.
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13.8 Priming Is Required to Start DNA Synthesis
All DNA polymerases are to elongate DNA chain but notto initiate DNA replication, and require a 3!OHpriming
end for DNA synthesis.
A molecule that provides a free 3-OH end is calledprimer.
Primers can be a short RNA synthesized by primase,nicked DNA, tRNA (retrovirus), or a protein (adenovirus).
Figure 13.10: A DNA polymerase requires a 3!OH end to initiate replication.
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13.10 DNA Polymerase HoloenzymeConsists of
Subcomplexes
The E. colireplicase DNA polymerase III(DNA pol III) isa 900 kD complex with a dimeric structure.
Each monomeric unit has a catalytic core, a dimerizationsubunit, and a processivity component.
DNA polymerase holoenzyme = core enzyme + clamp +clamp loader + tau (!).
Core enzyme = !(polymerase) + "(3-5 exonuclease) +#(stimulates exonuclease activity)
$clamp (sliding clamp): homodimers bind to DNA andcore enzyme"processivity factor.
"complex: composed of 5 proteins (", #, #, $, %), placesthe &clamp on DNA using ATP hydolysis.
!: links the two catalytic cores.
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13.10 DNA Polymerase Holoenzyme Consists of
Subcomplexes
Figure 13.14
Core
Clamploader
Clamp
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13.11 The Clamp Controls Association of Core Enzyme
with DNA
Figure 13.16: The helicase creating the replication fork is connected to two DNApolymerase catalytic subunits, each of which is held on to DNA by a sliding clamp.
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13.11 The Clamp Controls Association of Core Enzyme
with DNA
The core on the leading strand is processive because itsclamp keeps it on the DNA.
The clamp associated with the core on the lagging stranddissociates at the end of each Okazaki fragment andreassembles for the next fragment.
Figure 13.16: The helicase creating the replication fork is connected to two DNApolymerase catalytic subunits, each of which is held on to DNA by a sliding clamp.
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13.11 The Clamp Controls Association of Core Enzyme
with DNA
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13.11 The Clamp Controls Association of Core Enzyme
with DNA
The helicase DnaBisresponsible for interacting with
the primase DnaGto initiate
each Okazaki fragment.
Figure 13.17: Each catalytic core of Pol III synthesizes a
daughter strand. DnaB is responsible for forward
movement at the replication fork.
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13.11 The Clamp
Controls Association of
Core Enzyme with DNA
Figure 13.18: Core polymerase and
the &clamp dissociate at completion
of Okazaki fragment synthesis and
reassociate at the beginning.
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13.12 Okazaki Fragments
Are Linked by Ligase
Okazaki fragmentsynthesis: priming (RNA
primer synthesis by
primase), extension,
removal of RNA primer,gap filling, and nickligation.
Figure 13.19
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13.12 Okazaki Fragments Are Linked by Ligase
priming (RNA primer synthesis): DnaG (E. coli) and Pol '(eukaryote)
Extension: DNA pol III (E. coli) and Pol #+ Pol ((eukaryote) Removal of RNA primer:
E. coli: DNA pol I (5-3 exonuclease) Eukaryotes: RNase H (endonuclease specific for RNA:DNA hybrid)
and FEN1 (5-3 exonuclease)
Nick ligation: DNA ligase (E. coli) and DNA ligase I(eukaryotes)
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13.13 Separate Eukaryotic DNA Polymerases
Undertake Initiation and Elongation
Eukaryotic replication is similar to bacterial replication:semiconservative, bidirectional, and semidiscontinuous.
Eukaryotic genome has multiple replicons replicatingduring S phase of the cell cycle.
Three DNA polymerases are required for eukaryotic DNAreplication: pol !/primase, pol and pol ".
The DNA polymerase '/primase complex initiates thesynthesis of leading and lagging strands.
Pol !elongates the leading strand and Pol "elongatesthe lagging strand.
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13.13 Separate Eukaryotic DNA Polymerases
Undertake Initiation and Elongation
Figure13.23
DNA pol !/primasesynthesizes RNA (~10 nt)followed by 20-30 bases of
DNA (DnaG synthesizes RNA
only).
DNA pol 'is replaced by pol !on the lagging strand and by
pol (on the leading strand
polymerase switch.
Replication factor C (RFC,clamp loader) and proliferatingcell nuclear antigen (PCNA,
sliding clamp), and
minichromosome maintenance
(MCM, helicase) are required.
trimer
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13.13 Separate Eukaryotic DNA Polymerases
Undertake Initiation and Elongation
Figure 13.22: Similar functions are required at all replication forks.
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13.14 Lesion Bypass Requires Polymerase
Replacement
A replication fork stalls when it arrives at damaged DNA. Replicases are replaced by error-prone DNA
polymerases, which add random bases to allow bypass
the lesion"mutations are repaired after DNA
replication. Eukaryotes have 5 error-prone DNA pols and E. colihas
2 error-prone DNA pols.
13 14 L i B R i P l
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13.14 Lesion Bypass Requires Polymerase
Replacement
Figure 13.04
Figure 13.21
E. coliEukaryotes
Replicases: high-fidelity
Error-prone polymerases
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13.15 Termination of Replication
tersites:!E. coli DNA replicationtermination sequence.
!Contains a short, ~23bp sequence; 2 clusters
of 5 ter sites.
!Recognized by Tus,which prevents the
replication fork from
proceeding.
Unidirectional: fork 1 canpass ter B,C, F,G, and J
but for 2 cannot.Figure 13.27