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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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Figure 13.03

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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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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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Replication

Figure 13.05

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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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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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with DNA

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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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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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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

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