• Prokaryotic DNA Replication

DNA replication is perfomed by a

multienzyme complex >1 MDa
DNA
Nucleotides

Replisome:
DNA polymerases
Helicase
Primase
SSBs
DNA ligase
Clamps
(Topoisomerases)

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 Replication is semiconservative,
accurrate and fast

Accuracy
1 error in 1 billion
bases

Speed
500 nt/s in bacteria
50 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

3
 Polymerisation in detail
(dNMP)n + dNTP (dNTP)n+1 + PPi
DNA
2 Pi

Complementary basepairing and matching

hydrogen bonds is required
Incorrect basepairing

4
DNA is synthesized by DNA polymerase

DNA polymerase III is a protein complex

Subunit function
 not known
 3’ exonuclease
 polymerase
 clamp
 dimerisation

 clamp loader



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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 repair DNA repair Replication

RNA primer
removal

DNA polymerase I

Found by Arthur Kornberg, mid 1950’s

Three 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 site
3. 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 pairs

AT-rich
Initiation proteins bind to 9 bp consensus sequence

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 Inititation of replication
at the replication origin

Regulation of initiation of replication

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 DNA is synthesized in the replication fork
in 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 nt

Bind in clusters
Cooperative binding
Lowers 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 prokaryotes

ATP 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 forks

are synchronized by 10
23 bp Ter sequences that
bind Tus proteins

Tus proteins can only be

displaced by replisomes
coming 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 genome
Multiple linear chromosomes
Centromers
Telomeres
Histones

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 Lagging DNA repair Mitochondria Lagging Leading

strand l DNA strand strand
primer replication replication replication
synthesis

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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 only

during S phase and are added
as replication proceeds

Some histone parts are

”inherited” some are new

The spacing of histones every

200 nt might be the reason for
the shorter Okazakifragments
in 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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