DNA Replication The basic rules for DNA replication DNA synthesis at the replication fork Termination of replication Other modes of DNA replication DNA Polymerases Initiation of replication Regulation of re-initiation
Jan 17, 2016
DNA Replication
The basic rules for DNA replication
DNA synthesis at the replication fork
Termination of replication
Other modes of DNA replication
DNA Polymerases
Initiation of replication
Regulation of re-initiation
Initiation of replication
Common features of replication origins
Common events of initiation
Priming
(proposed by F. Jacob, S. Brenner and J. Cuzin, 1963)
All the DNA replicated from a particular origin as a replicon.
Binding of the initiator to the replicator stimulates initiation of replication.
The replicon model of replication initiation:
Replicator
Replicator: the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of DNA replication (*the origin of replication is part of replicator)
Initiator
Initiator: the DNA-binding protein that specifically recognizes a DNA element in the replicator and activates the initiation of replication
Initiator binding siteEasily melted region
Replicator
InitiatorDNA binding
DNA unwinding
Protein recruitment
Priming and DNA synthesis
Th
e f
un
cti
on
s o
f in
itia
tor
(Origin)
DNA binding
DNA unwinding
Protein recruitment
Priming and DNA synthesis
oriCDnaA
DnaB/DnaC
E. coli
oriC : the origin of replication in E. coli
Easily melted(AT rich)
Initiator (DnaA) binding site
Figure 14.26
Consensus sequence
GATCTNTTNTTTTCTAGANAANAAAA
Consensus sequence
TTATNCANAAATANGTNT
L, M, R repeats (13 bp) 1~4 repeats (9 bp)
oriC:
The replicator (origin) of S. cerevisiae:
圖引用自: Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17
Origin recognition complex (ORC)
(Initiation complex)
ACS: ARS consensus sequence
ARS (Autonomously replicating sequence)
~ 150 bp
Mutation in ARS
Mutations in B elements reduce origin function.
Mutations in core consensus abolish origin function.
Fig
ure
13.2
0
DNA binding
DNA unwinding
Protein recruitment
Priming and DNA synthesis
oriCDnaA
DnaB/DnaC
E. coli
Fig
ure
14.2
7
Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-26.
DNA helicase(DnaB)
DNA helicaseLoader (DnaB)
DnaA.ATP
DnaAATP
HU + ATP
(histone like protein)
DNA bending
Super-helical tension
Strand separation
DNA helicase(DnaB)
DNA helicaseLoader (DnaB)
Fig
ure
14.1
0
Two types of function are needed to convert dsDNA to the single-stranded state:
2. Single-strand binding proteins bind to the ssDNA, preventing it from reforming the duplex state
1. Helicases separate the strands of DNA, usually using the hydrolysis of ATP to provide the necessay energy
Single-strand binding proteins
DNA helicase(DnaB)
DNA helicaseLoader (DnaB)
Primase
For DNA replication, a primase is required to catalyze the synthesis of RNA primer.
Primase in E. coli:
An RNA polymerase
Encoded by the dnaG gene
Synthesizing short stretches of RNA
Fig
ure
14.1
4
Protein required to initiate replication at the E. coli origin:
Protein Function
DnaA protein Recognizes origin sequence; open duplex
at specific sites in origin
DnaB protein (helicase) Unwinds DNA
DnaC protein Required for DnaB binding at origin
HU DNA bending protein; stimulates initiation
Primase (DnaG protein) Synthesizes RNA primers
Single-strand DNA-binding protein (SSB)
Binds single-strand DNA
DNA gyrase
(DNA topoisomerase)
Relieves torsional strain generated by DNA
unwinding
Dam methylase Methylates 5’-GATC-3’ sequences at oriC
Summary
DNA Replication
The basic rules for DNA replication
DNA synthesis at the replication fork
Termination of replication
Other modes of DNA replication
DNA Polymerases
Initiation of replication
Regulation of re-initiation
DNA synthesis at the replication fork
Proteins at the replication forks
Coordinating synthesis of the lagging and leading strands
in eukaryotic cells
in E. coli
DNA replication is semidiscontinuous.
Figure 14.9
1000-2000 nt in prokaryotes100-400 nt in eukaryotes
Okazaki fragments
*SSB: Single-strand binding proteins
Topoisomerase
HelicasePrimase
SSB
DNA replicase(DNA polymerase)
Primosome
Proteins required at the replication forks:
Primer removal enzyme
DNA ligase
Different replicase units are required to synthesize the leading and lagging strands.
In E. coli both units contain the same catalytic subunit of DNA Pol III. In other organisms, different catalytic subunits may be required for each strand.
The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits.
Each polymerase catalytic subunit is held on DNA by a sliding clamp.
Figure 14.19
The polymerase that synthesizes the lagging strand dissociates at the end of Okazaki fragment and then reassociates with a primer in the single- stranded template loop to synthesize the next fragment.
The polymerase that synthesizes the leading strand moves continuously.
Figure 14.19
In E. coli: DnaB
DnaGDNA Pol III
holoenzyme
’
E. coli DNA Polymerase III holoenzyme
Based on Figure 14.17
Core enzyme
proofreading
polymerizationpolymerization
Sliding
clamp
’
Clamp loader
Core enzyme dimerization
Sliding
clamp
’
Clamp loader
ATP
’ATP
’ADP
’ATP
Sliding clamp
ATP
hydrolysis
Pi
Core enzyme: 10 ~ 15
Holoenzyme: >500000
converts Pol III from a distributive enzyme to a highly processive
enzyme.
Processivity
Figure 14.18
’
subunits maintain dimeric structure of Pol III and interact with DnaB
DnaB(helicase)
Lagging strand synthesis
Leading strand
synthesis
Each catalytic core of polymerase III synthesizes a daughter strand.
DnaB (helicase) is responsible for forward movement at the replication fork.
Figure 14.20
What happens to the loop when the Okazaki fragment is completed?
Figure 14.21
Initiation of
Okazaki fragment
Termination of
Okazaki fragment
Dissociation of core
and clamp
Reassociation of clamp
1
2 3
4
Figure 14.20
5. Reassociation of core
Each Okazaki fragment is synthesized as a discrete unit.
Primase synthesizes RNA primer.
DNA Pol III extends primer into Okazaki fragment.
Next Okazaki fragment is synthesized.
Leading strand
Lagging strand
Okazaki fragments are linked together.
DNA Pol I uses nick translation to replace RNA primer with DNA.
Ligase seals the nick.
Figure 14.22
5’3’3’ 5’
RNA primer
5’3’ Exonuclease activity of DNA Pol I:
Figure 14.5
Ligase NH3+
Ligase NH2
+P
O
O-
O Ribose Adenine
+
Adenylylation of DNA ligase
P
O
O-
O Ribose AdenineOR
AMP
NAD+ (R = NMN)or ATP (R = PPi)
11
Mechanism of the DNA ligase reaction
+ NMN (or PPi)
Ligase NH2
+P
O
O-
O Ribose Adenine
Ligase NH3+
Activation of 5’ phosphate in
nick
22
33
Fig
ure
14.2
3
*
PO O-
O
O
PO O-
O
RiboseAdenine
HO●●
The 3’-hydroxyl group attacks the phosphate and displaces AMP, producing a phosphodiester bond.
3’
33
Pol/primase
Eukaryotic cellEukaryotic cell
(RFC)
PCNA
Pol
Pol
Eukaryotes have many DNA polymerases.
Fig
ure
14.2
4
Eukaryotic DNA polymerases for replication in nucleus:
DNApolymera
se
Primaseactivity
Processivity
Proof-reading
Function
+ moderate - Primer synthesis
- High +
Leading/lagging? strand
synthesis
- High +laggingstrand
synthesis
DNA polymerase Pol/primase):
2 subunits: Pol
2 subunits: primase
DNA synthesis
RNA synthesis
3’ 5’
RNA DNA (iDNA)
5’ 3’OH
~ 10 bp 20-30 bp
DNA polymerase switching during eukaryotic DNA replication:
DNA Pol / primase RNA primer synthesis
by primase
DNA synthesis by Pol
P
RNA
iDNA Wats
on,
J. D
. et
al. (
20
04
) M
ole
cula
r B
iolo
gy o
f th
e g
ene.
5th
ed.
CSH
L Pre
ss.
Fig.
8-1
6.
Slidingclamp Pol/primase
DNA Pol (or
iDNA
*
R-FC binds to the 3’ end of iDNA and displaces pol /primase
PCNA binds pol or
RF-C: Clamp loader
PCNA: Sliding clamp
*
R-FC attracts PCNA
PCNA- Proliferating cell nuclear antigen
:
圖引用自: Voet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-1
(trimer)
Proteins required at the replication forks:
Summary
DNA topoisomerases are also required!
Fig
ure
14.2
5
There are two ways to think of the relative motion of the DNA and replication machinery:
1. The replication machinery moves along
the DNA. (similar to a train moving along its
track)2. The DNA moves while the replication machinery is static. (similar to film moving into a movie projector)
The two replisomes of E. coli are linked together and tethered to one point on the bacterial inner membrane.
Helicases(double-hexamers)
Pol III holoenzyme
Pol III holoenzyme
圖引用自: Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a
Origin
TerminatorChromosome
Replisomes
Replication begins
Origins separate
Cell elongates as replication continues
Chromosomes
separate
Cells divid
e
圖引用自: Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a