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• Prokaryotic DNA Replication

DNA replication is perfomed by a multienzyme complex >1 MDa

DNANucleotides

Replisome:DNA polymerasesHelicasePrimaseSSBsDNA ligaseClamps(Topoisomerases)

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Replication is semiconservative, accurrate and fast

Accuracy1 error in 1 billion bases

Speed500 nt/s in bacteria50 nt/s in mammals

Each original strand functions as template for DNA synthesis

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After each replication cycle, DNA is doubled

DNA is synthesized in 5´to 3´direction

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Polymerisation in detail

(dNMP)n + dNTP (dNTP)n+1 + PPi

DNA

2 Pi

Complementary basepairing and matching hydrogen bonds is required

Incorrect basepairing

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DNA is synthesized by DNA polymerase

DNA polymerase III is a protein complex

not known 3’ exonuclease polymerase clamp dimerisation clamp loader

Subunit function

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E. coli contains multiple DNA polymerases

DNA pol I DNA pol II DNA pol III

Number/cell 400 100 10

Speed (nt/s) 16-20 2-5 250-1000

3´exonuclease Yes Yes No

5´exonuclease Yes No No

Processivity 3-200 10 000 500 000

Role DNA repairRNA primer removal

DNA repair Replication

DNA polymerase I

Found by Arthur Kornberg, mid 1950’sThree enzymatic activities:• Polymerase activity• 3’ to 5’ exonuclease activity• 5’ to 3’ exonuclease activity

Klenow enzyme is lacking one subunit responsible for the 5’ to 3’ exonuclease activity

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DNA polymerase requires

1. A free 3’-OH group supplied by RNA Primer for start of polymerisation

2. Mg2+ ions for activity in active site3. A template to copy

DNA replication initate at origin of replication

Bacterial chromosome doubles in 40 min

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DNA replication is bidirectional

The replication origin OriC in E.coli

245 base pairsAT-richInitiation proteins bind to 9 bp consensus sequence

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Inititation of replicationat the replication origin

Regulation of initiation of replication

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DNA is synthesized in the replication forkin 5’ to 3’ direction

Leading strand synthesis is continuous whereas lagging strand is synthesized in

fragments

Length of Okazaki fragments in prokaryotes are 1000-2000 nt,in eukaryotes 100-200 nt

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Mistakes during DNA synthesis are edited

This results in a very low error rate of 1 in 1 billion nucleotides

3’ to 5’ exonuclease activity corrects errors

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Requirements for proofreading mechanism

• Addition of nucleotides to RNA primer

• Absolute requirement for a match at the 3’ end of the extended strand

• 3’ to 5’ exonuclease activity of DNA polymerase

• Template DNA is identified by methylation (E. coli) or absence of nicks (eukaryotes)

5’ to 3’ exonuclease activity causes strand displacement/nick translation

No net synthesis

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Helicase unzips double-helix

Single strand binding proteins keep strands single stranded

Each SSB bind to 7-10 ntBind in clustersCooperative bindingLowers Tm of template

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Binding of SSBs to DNA

DNA pol. is attached to strand by Clamp loader and Sliding clamp

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Sliding clamp

Accounts for high processivity:Limits association and dissociation

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DNA primase

Makes the 10 nt RNA primer required for start of replication

In beginning of each Okazaki-Fragment

RNA primer is later erased and replaced with DNA by DNA pol I

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DNA ligase

Seals the nicks between Okazaki fragments

Requires close and free 3’-OH and 5’-P and proper base-pairing

NAD+ required in prokaryotesATP required in eukaryotes

Nick sealing by DNA ligase

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Topoisomerases

Relieves torsional stress caused by rotation of DNA ahead of the fork

10 nucleotides = 1 turn

Topoisomerase I

Breaks one strand of the duplex

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Mechanism of topoisomerase I

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Topoisomerase II(DNA gyrase)

Breaks both strands of the duplex

Introduces negative superhelices

ATP dependent

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Summary of replication

DNA is bent duing replication process

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DNA is proofread during the process

Termination of replication

The two replication forksare synchronized by 1023 bp Ter sequences that bind Tus proteins

Tus proteins can only be displaced by replisomescoming from one direction

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Resolvation of replication products by decatenation

• Eukaryotic DNA Replication

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Eukaryotes has some special features

Larger genomeMultiple linear chromosomesCentromersTelomeresHistones

DNA replication

DNA replication takes place during the S phase part of the interphase of the cell cycle. S for synthesis. Two identical copies of the chromosome are produced, attached at the centromer.

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Parts on the yeast chromosome contain

Autonomous Replicating Sequence

Eukaryotes also contain multiple DNA polymerases

DNA pol DNA pol DNA pol DNA pol DNA pol

3´exonuclease No No Yes Yes Yes

Fidelity 10-4 - 10-5 5x10-4 10-5 10-5 - 10-6 10-6 - 10-7

Processivity Moderate Low High High High

Role Laggingstrand primer synthesis

DNA repair Mitochondrial DNA replication

Laggingstrand replication

Leadingstrand replication

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Inititiation of replication in eukaryotes

Due to the eukaryotic chromosome size, multiple replication origins are needed• Eukaryotic replication origins are organized in replicons, 20-80 ori/cluster• Replication is initated all through the S phase• Active chromatin replicate early, condensed chromatin replicate late• A replication bubble is formed at each ori, forks moving in both directions• Each ori is only replicated once

Histones are synthesized onlyduring S phase and are addedas replication proceeds

Some histone parts are ”inherited” some are new

The spacing of histones every 200 nt might be the reason forthe shorter Okazakifragmentsin eukaryotes and the slower speed of replication

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New histones are modified

Telomerase recognizes the G-rich 3’- end of the chromosome (telomere)

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Comparison prokaryotic vs eukaryotic replication

Prokaryote (E.coli) Eukaryote (Human)

# Origins of replication 1 1000-10000 in replicons

Speed of replication 500 nt/s 50 nt/s

Time for replication 40 min 8 hours

Okazaki fragments 1000-2000 nt 100-200 nt

Polymerases 3 (5) 5 (10)

Chromosomes 1, circular 46, linear

Other Telomeres, histones

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• Reverse transcription

Retroviruses are mobile genetic elements

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RNA-dependent DNA polymerase

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