• 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)
1 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
2 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
5 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
6 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
7 DNA replication is bidirectional
The replication origin OriC in E.coli
245 base pairs AT-rich Initiation proteins bind to 9 bp consensus sequence
8 Inititation of replication at the replication origin
Regulation of initiation of replication
9 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
10 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
11 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
12 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
13 Binding of SSBs to DNA
DNA pol. is attached to strand by Clamp loader and Sliding clamp
14 Sliding clamp Accounts for high processivity: Limits association and dissociation
15 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
16 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
17 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
18 Mechanism of topoisomerase I
19 Topoisomerase II (DNA gyrase)
Breaks both strands of the duplex Introduces negative superhelices ATP dependent
20 Summary of replication
DNA is bent duing replication process
21 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
22 Resolvation of replication products by decatenation
• Eukaryotic DNA Replication
23 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.
24 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
25 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
26 New histones are modified
Telomerase recognizes the G-rich 3’- end of the chromosome (telomere)
27 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
28 • Reverse transcription
Retroviruses are mobile genetic elements
29 RNA-dependent DNA polymerase
30 31