• 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
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
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 OkazakiFragment
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
strand
primer
synthesis
DNA repair
Mitochondria Lagging
l DNA
strand
replication
replication
Leading
strand
replication
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