Plant Molecular and Cellular Biology
Lecture 4: E. coli DNA Replicase
Structure & Function
Gary Peter
Learning Objectives
1. List and explain the
mechanisms by which E.
coli DNA is replicated
2. Describe and explain the
structure and functions of
the enzymes and their
subunits that replicate DNA
in E. coli
Wang et al., Nucleic Acids Res. 2004; 32(3): 1197–1207
Processivity
z
z
The number of
nucleotides added
during each binding
and release from the
primed template
The ability of the DNA
polymerase to remain
associated with the
DNA template
z
Typical processivity of
enzymes in vitro
z
z
z
z
Klenow 50-60 nt
T7 300 nt
Taq 22 nt
Pfu 6.4 nt
Strand Displacement Activity
z
Strand Displacement:
z
The ability to displace
downstream DNA
encountered during
synthesis.
z
Protocols such as the
isothermal amplification
method Strand
Displacement
Amplification (SDA)
exploit this activity.
z
When new synthesis
starts at a nick it
displaces a strand.
The displaced strand
then itself becomes a
template for the
synthesis of a new
strand.
Strand Displacement &
Processivity of Bacteriophage
Phi29 DNA Polymerase
z
z
z
This polymerase has excellent strand
displacement activity and high
processivity and is used in strand
displacement amplification (SDA)
High displacement is likely due to a
tunnel that is too small for dsDNA to
enter and requires/induces strand
separation
The high processivity is likely due to
topological encirclement of both the
downstream template and the
upstream dsDNA
z
This structure abrogates the need for
ancillary factors such as helicase and the
clamp
Kamtekar et al., 2004 Mol. Cell 16 (4): 609-618
Functionality of
Various DNA
Polymerases
3'->5'
Proofreading
Strand
Displacement
Primary
Applications
Mesophilic DNA Polymerases
phi29 DNA
Polymerase
++++
+++++
Strand
Displacement
Applications
+++++
-
Polishing Ends,
2nd Strand
Synthesis
DNA Polymerase I
++
-*
Nick Translation
DNA Polymerase I,
Klenow Fragment
++
++
Polishing Ends
Klenow Fragment (3'
-> 5' exo-)
-
+++
Labeling
T7 DNA Polymerase
(unmodified)
++++
-
Terminal Transferase
-
NA
T4 DNA Polymerase
*Degrades displaced strand
Site Directed
Mutagenesis
3' terminal
Tailing
http://www.neb.com/nebecomm/tech_referenc
e/polymerases/polymerases_from_neb.asp
3'->5'
Proofreading
Strand
Displacement
Primary
Applications
Mesophilic DNA
DNA Polymerases
Polymerases
hermophilic
Phusion™ High
Fidelity DNA
Polymerase
Phusion™ Hot
Start High Fidelity
DNA Polymerase
+++++
+++++
-
PCR (high
fidelity)
-
Hot Start PCR
(high fidelity)
DyNAzyme™ EXT
DNA Polymerase
+
+
PCR (difficult or
long)
DyNAzyme™ II
Hot Start DNA
Polymerase
-
-
PCR (hot start)
Taq DNA
Polymerase
-
-*
PCR (routine),
Primer Extension
+++
++
PCR (high
fidelity), Primer
Extension
VentR (exo-) DNA
Polymerase
-
+++
PCR,
Sequencing
Deep VentR DNA
Polymerase
+++
++
PCR (high
fidelity), Primer
Extension
Deep VentR (exo-)
DNA Polymerase
-
+++
PCR (long),
Primer Extension
9°Nm DNA
Polymerase
+
+++
Primer Extension
Therminator DNA
Polymerase
-
+
Chain Terminator
Applications
Bst DNA
Polymerase, Large
Fragment
-
+++
VentR DNA
Polymerase
Strand
Displacement
Applications
3'->5'
Proofreading
Strand
Displacement
Primary
Applications
Mesophilic
DNA Polymerases
Other
Polymerases
M-MuLV
Reverse
Transcriptase
-
+++
cDNA
Synthesis
AMV
Reverse
Transcriptase
-
+++
cDNA
Synthesis
E. coli
Poly(A)
Polymerase
-
NA
3´ labeling
of RNA
Overview of Basic Steps in
DNA Replication
1.
2.
3.
4.
5.
Unwinding of the DNA strands
Recruitment of DNA polymerase complex &
auxiliary factors
Initiation of new chain
Elongation of the new chain by addition of
mononucleotides
Covalent closure of the new chains to form
one new DNA molecule
Standard Biochemical Approach to Identify
and Characterize the Proteins/Enzymes
that Mediate a Specific Process
z
z
z
z
z
z
Identify proteins involved
Determine the stiochiometry of the subunits
Determine the structure and function(s) of the
subunits
Determine the spatial arrangement of the
subunits
Determine the dynamics and steps in the
reaction each one mediates
Determine the regulation
Prokaryotic Replication Fork
z
z
z
Leading strand (5’>3’)
Lagging strand (3’>5’)
Enzymes
z
z
z
z
z
z
DNA primase
DNA helicase
Single strand binding
proteins
DNA ligase
DNA polymerases
Topoisomerases
Replisome
Close Association of Proteins
into a Replisome at the Fork
z
z
DNA polymerase III
holocomplex
Primosome
z
DNA helicase and DNA
primase located at the
center of the fork where
the two strands of the
helix are unwinding
bound to DNA pol III
Model for the Spatial Organization
of the the Replisome
2003 Molecular Microbiology, 49, 1157–1165
DNA Polymerase III Holoenzyme
z
A holoenzyme is the
fully functional form of
an enzyme which
contains all of the
necessary subunits to
be fully active
z
DNA Polymerase
Holoenzyme
z
z
z
Core enzyme
The sliding clamp
Clamp loading
complex
Comparison of DNA
polymerases I and III
Structure
Activities
Vmax (nuc./sec)
Processivity
Molecules/cell
DNA polymerase III
DNA polymerase I
DNA Pol III holoenzyme is an asymmetric dimer; i. e.,
two cores with other accessory subunits. It can thus
move with the fork and make both leading and
lagging strands.
DNA Pol I is a monomeric protein with three active
sites. It is distributive, so having 5'-to-3'
exonuclease and polymerase on the same
molecule for removing RNA primers is effective and
efficient.
Polymerization and 3'-to-5' exonuclease, but on
different subunits. This is the replicative polymerase
in the cell. Can only isolate conditional-lethal dnaE
mutants. Synthesizes both leading and lagging
strands. No 5' to 3' exonuclease activity.
Polymerization, 3'-to-5' exonuclease, and 5'-to-3'
exonuclease (mutants lacking this essential activity
are not viable). Primary function is to remove RNA
primers on the lagging strand, and fill-in the
resulting gaps.
250-1,000 nucleotides/second. This is as fast as the
rate of replication measured in Cairns' experiments.
Only this polymerase is fast enough to be the main
replicative enzyme.
20 nucleotides/second. This is NOT fast enough to
be the main replicative enzyme, but is capable of
"filling in" DNA to replace the short (about 10
nucleotides) RNA primers on Okazaki fragments.
Highly processive. The beta subunit is a sliding
clamp. The holoenzyme remains associated with the
fork until replication terminates.
10-20 molecules/cell. In rapidly growing cells, there
are 6 forks. If one processive holoenzyme (two
cores) is at each fork, then only 12 core polymerases
are needed for replication.
Distributive. Pol I does NOT remain associated
with the lagging strand, but disassociates after
each RNA primer is removed.
About 400 molecules/cell. It is distributive, so the
higher concentration means that it can reassociate
with the lagging strand easily.
http://oregonstate.edu/instruct/bb492/lectures/DNAII.html
DNA Polymerase III –Core Enzyme
Structure
z
z
z
A heterotrimer of the 3 subunits with different
functions in a 2:2:2 stiochiometry
α subunit is the DNA polymerase with sequence
similarity to C family polymerases
No crystal structure exists for this polymerase
DNA Polymerase III –Core Enzyme
Function
z
z
The core complex can catalyze DNA synthesis
(20 nt/s)
Without ε subunit the enzyme is not highly
processive 1500 nt with each binding and
release
Presence of e stimulates processivity – this helps
insure the fidelity as higher rates of DNA synthesis
have the proofreading activity
z
Subunit
Function
α
5’-3’ DNA polymerase activity- no proofreading activity (8 nt/s)
ε
3’-5’ proofreading exonucelase activity
θ
Stimulates proofreading exonuclease (not an essential gene)
DNA Polymerase III –β sliding
clamp: Structure
z
z
z
Interacts with the α
subunit of the DNA
polymerase
3 domains
Assembles into a
dimer with a circular
structure and 35
angstrom diameter
hole in the middle
where DNA is bound
Sliding Clamp of DNA
Polymerase: Function
z
z
Increases the rate of
DNA synthesis (750
ntd/s)
Confers extended
processivity to the
DNA polymerase
(>50 kb).
z
z
z
a) The γ complex clamp loader associates
tightly with β when bound to ATP. DNA
triggers ATP hydrolysis, resulting in low
affinity for β and DNA.
(b) When Pol III, the replicative polymerase,
encounters a lesion in the DNA template, it
stalls, unable to overcome its inherent
fidelity to incorporate opposite a damaged
base. Stalling allows an error-prone
polymerase, such as Pol IV (red) passively
traveling on β, an opportunity to trade places
with Pol III on β to replicate past the lesion.
[Adapted with permission from (135).]
(c) Pol III maintains a tight grip on β via the
polymerase C terminus. However, when it
completely replicates its substrate DNA, the
polymerase must release from β to recycle
to the next primed site. The τ subunit
modulates this interaction, binding the
polymerase C tail only when no more singlestranded template is present. This severs
the connection between the polymerase and
the clamp
DNA Polymerase III – The Clamp
loading Complex Structure
z
The clamp loader is
composed of 5
subunits that are
essential for its
function and 2
subunits that link it
to SSB and primase
Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315
DNA Polymerase III – The Clamp
loading Complex Function
•The γ complex uses the energy of ATP
binding and hydrolysis to topologically link
β to a primed DNA, then it ejects from
DNA, leaving the closed clamp behind.
•The three γ subunits bind ATP and are the
"motor" of the complex.
•The δ subunit is the "wrench" because it
is the main β clamp-interacting subunit,
and it can open the dimer interface by
itself.
•The δ' subunit modulates δ-β contact and
is a rigid protein, which remains stationary
while other parts move.
•The χ and ψ subunits are not essential for
the clamp-loading mechanism, but
•χ links the clamp loader to SSB and
primase
•ψ connects χ and strengthens the γ3δδ'
complex
DNA Primase Function & Activity
z
De novo 5’>3’ synthesis
of short,~10 nucleotide
long RNA strands
z
z
Leading strand synthesis
only one RNA primer
Lagging strand synthesis
z RNA primer laid down
every ~ 100-200
nucleotides
DNA Primase: Structure
z
There are three functional domains in the protein.
z The N-terminal 12 KDa fragment contains a zinc-binding motif.
z The central fragment of 37 KDa contains a number of conserved
sequence motifs that are characteristic of primases, including the socalled "RNA polymerase (RNAP)-basic" motif that shows homology with
equivalent motifs in prokaryotic and eukaryotic RNAP large subunits.
This suggests that primases might share a common structural
mechanism with RNAP.
z The C-terminal domain of approximately 150 residues is the part of the
protein responsible for interaction with the replicative helicase, DnaB, at
the replication fork.
DNA Helicases:
Function &
Activities
z
z
Unwinding the dsDNA at the
replication fork for DNA
replication, transcription,
repair, recombination
ATPase activity used for DNA
strand unwinding and
movement along single
stranded DNA
z
z
z
Two different helicases with
the ability to move in opposite
directions (5’>3’ & 3’>5’)
ATP hydrolysis is stimulated by
single stranded DNA
Helicases move at rates up to
1000 nucleotides/sec
DNA Helicase: Structure
z
z
Hexameric structure
with 6 identical subunits
Loading onto DNA
occurs through the help
of loading proteins
which promote
assembly of the
hexamers around the
DNA
Leading vs. Lagging Strand
Synthesis
z
z
z
Leading
Highly processive
Polymerase moves 5’-3’
Strand displacement is
due to the joint action of
polymerase III, rep
protein and HDP
z
z
z
z
z
z
Lagging
Short fragments
Polymerase moves 3’-5’
Primase to polymerase
switching occurs rapidly
Single stranded binding
proteins more important
DNA polymerase I
involvement
Elevated DNA ligase
involvement
Single Stranded Binding
Proteins: Function & Activities
z
z
Involved with DNA
replication,
recombination, repair
Stabilizes ssDNA upon
binding to the single
strands after the helix is
opened by helicases
Single Stranded Binding Proteins
Structure of E. coli SSB
z
z
z
Stable tetrameric organization
DNA binding domain makes extensive contacts with ssDNA
Two forms of cooperative binding
z
At low monovalent salt concentrations (<10 mM NaCl and high protein to DNA ratios,
Eco SSB displays ‘unlimited’ cooperative binding to long ssDNA, resulting in the
formation of long protein clusters. However, at high salt concentrations (> 0.2 M NaCl
or > 3 mM MgCl2 and low protein binding density, Eco SSB binds to single stranded
polynucleotides in a ‘limited’ cooperativity mode, in which the protein does not form
long clusters along the ssDNA
Lagging Strand
Synthesis
z
Replication Fork
z
z
z
z
z
z
DNA polymerase III
Primosome
SSB
Rnase H
DNA polymerase I
DNA ligase
The primase-to-polymerase switch
during lagging strand synthesis
z
z
z
(A) DnaB helicase encircles the lagging
strand and primase has synthesized a
primer. The holoenzyme consists of a dimer
of tau that binds two polymerase cores, one
gamma complex clamp loader, and two beta
clamps. Tau and primase interact with
DnaB. Primase must contact SSB to remain
on the RNA primer. (B) The chi subunit of
gamma complex interacts with SSB,
severing the primase-SSB contact and
resulting in primase displacement. (C)
Primase is then free to synthesize another
RNA primer upon contact with DnaB.
(B) also shows that the lagging strand
polymerase releases the beta clamp and
DNA upon finishing an Okazaki fragment.
(C) shows that after gamma complex
assembles the new beta clamp on the
upstream primer, the lagging polymerase
recruits the new beta clamp (shaded dark)
assembled on the upstream RNA primer for
the next Okazaki fragment.
http://oregonstate.edu/instruct/bb492/figletters/FigU.html
The Winding Problem
z
The parental DNA winds
tightly ahead of the
replication fork
z
z
z
In E. coli the replication fork
travels at 500 bp/sec
Every 10 bp replicated is 1
turn of the DNA helix and
the helix ahead of the fork
becomes wound tighter
(48 revolutions/sec)
Solution is provided by DNA
topoisomerases
z
z
These enzymes release the
tightly wound DNA
They can also release the
two new DNAs after
replication is completed
DNA Topoisomerase I
z
Produces a transient single
stranded break in the
phosphodiester backbone that
allows the two sections of the DNA
helix on each side of the break to
rotate freely thereby releasing the
tension built up from unwinding
PNAS 2003 100: 10629–10634
DNA Topoisomerase II
Summary
z
z
z
The enzymes that conduct DNA replication in E. coli are
organized into a replisome that contains two copies of
DNA polyermase III which act in concert synthesizing the
new strands on both the leading and lagging strands
Leading strand synthesis occurs very processively, in
contrast lagging strand synthesis involves multiple short
strand synthesis and the involvement of DNA
polymerase I, SSB, primase and DNA ligase more
prominently
DNA helicase unwinds the duplex ahead of the
replication fork and DNA topoisomerases relieve the
supercoiling tension introduced by helicase