• Prokaryotic DNA Replication
DNA replication is perfomed by a multienzyme complex >1 MDa DNA Nucleotides Replisome: DNA polymerases Helicase Primase...
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
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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 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 RNA primer removal
DNA repair
Replication
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 OkazakiFragment
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 strand primer synthesis
DNA repair
Mitochondria Lagging l DNA strand replication replication
Leading strand 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 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)
