Plant Molecular and Cellular Biology
Lecture 5: DNA Replicase Structure
& Function
Gary Peter
Learning Objectives
1. List and explain the
mechanisms by which
eukaryote DNA is
replicated
2. Describe and explain the
structure and functions of
the enzymes and their
subunits that replicate DNA
in eukaryotes
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 ε 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
Each subunit has 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).
Dynamics of the
Sliding Clamp
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. –DNA dependent
hydrolysis
B) Pol III is unable to overcome its inherent
fidelity to incorporate opposite a damaged
base at a lesion in the DNA template, and it
stalls. Stalling allows an error-prone
polymerase, such as Pol IV that passively
travels on β, the time to switch places with
Pol III on β and replicate past the lesion.
C) Pol III binds tightly to β via the its C
terminus. However, when replication is
complete, the polymerase must release from
β to rebind to the next primed site. The τ
subunit modulates this interaction, binding
the polymerase C tail only when no more
single-stranded template is present,
severing the connection between the
polymerase and the clamp
Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315
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
Assembly of the β Sliding
Clamp
z
The nucleotide free clamp
loader has low affinity for
the clamp
z
z
δ binds tight to β probably
sequestering or blocking
the other subunits without
ATP
When ATP binds the γcomplex changes
conformation and δ opens
one dimer interface
z
z
z
The clamp-clamp loader
complex has a high affinity
for primed DNA-templates
The DNA activates the
ATPase activity and allows
ring closure and ejection of
the loader complex
After ATP hydrolysis, the
clamp loader has a low
affinity for DNA until ADP
dissociates and the γ
complex binds ATP
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 10-15 bp 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 protein 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
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
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
Dynamic Organization of the Replisome:
Lagging Strand Synthesis
z
z
Lagging-strand replication is a discontinuous and starts
that repeats every 1–3 s. Each Okazaki fragment is
initiated by primase, which synthesizes an RNA primer
of about 10–12 nucleotides. Primase action requires
interaction with DnaB, which involves a C-terminal
region of primase. Primase extends the RNA in the
opposite direction of helicase unwinding and is
presumed to separate from DnaB, which may account
for its observed distributive action. Primase remains
attached to the RNA primed site through its interaction
with SSB. Although primase eventually dissociates,
release of primase is accelerated by the χ subunit of
the clamp loader, which binds SSB in a competitive
fashion, recruiting the clamp loader to the DNA
template to compete with primase. The clamp loader
then places β onto the primer for the lagging-strand
polymerase.
As the lagging polymerase extends a fragment, a loop
is generated because it is connected to the leading
polymerase (via the clamp loader), yet extends DNA in
the opposite direction. The 1–3 kb Okazaki fragment
will be completed within a few seconds , and at this
point, the core must rapidly release from DNA to start
the next fragment . The highly processive Pol III
requires a specific mechanism for this release step,
which disengages core from β, leaving the β clamp
behind on the finished fragment. The release step
occurs only at a nick, thus ensuring completion of the
fragment, and requires the τ subunit . The laggingstrand core is now free to bind a new β clamp placed
on the next RNA primer by the clamp loader.
Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315
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
Clamp Protein Conservation
E. Coli β clamp
Yeast PCNA
Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315
Conservation of the Clamp
Loader Structure
z
Structural comparison
of the Pfu clamploading complex with
the E. coli clamp loader
{gamma} complex and
the yeast clamp loaderclamp binary complex
E. coli
Pfu
Pfu
yeast
Miyata, Tomoko et al. (2005) Proc. Natl. Acad. Sci. USA 102, 13795-13800
Replisome
component
RFCa
Saccharomyces
cerevisiae (kDa)
RFC (277.7)a
RFC1 (94.9)
RFC2 (39.7)
RFC3 (38.2)
RFC4 (36.1)
RFC5 (39.9)
PCNA (28.9)
Human (kDa)
Function and remarks [Schizosaccharomyces pombe name (S.p.)]
RFC (314.9)a
p140 (128.2)
p37 (39.2)
p36 (40.6)
p40 (39.7)
p38 (38.5)
PCNA (28.7)
Pentameric clamp loadera
Binds ATP; phosphorylated
Binds ATP
Binds ATP
Binds ATP
Binds ATP or ADP
87 kDa homotrimeric processivity sliding clampa
Pol δ (220.2)a
Pol3 (124.6)
Pol δ (238.7)a
p125 (123.6)
Replicative DNA polymerasea
DNA polymerase, 3'-5' exonuclease, binds PCNA; subunit A (S.p. Pol3)
Pol31 (55.3)
p50 (51.3)
Structural subunit; subunit B (S.p. Cdc1)
Pol32 (40.3)
p66 (51.4)
Binds PCNA; subunit C (S.p. Cdc27); binds Pol α large subunit
—
p12 (12.4)
Structural, stimulates processivity; subunit D (S.p. Cdm1)
Pol ε (378.7)a
Pol2 (255.7)
Dpb2 (78.3)
Pol ε (350.3)a
p261 (261.5)
p59 (59.5)
Replicative DNA polymerasea
DNA polymerase, 3'-5' exonuclease (S.p. Pol2/cdc20)
Binds polymerase subunit (S.p. Dpb2)
Dpb3 (22.7)
Dpb4 (22.0)
p17 (17.0)
p12 (12.3)
Binds Dpb4
Present in ISW2/yCHRAC chromatinremodeling complex (S.p. Dpb4)
Pol αa
Pol α (355.6)a
Pol1 (166.8)
Pol12 (78.8)
Pri2 (62.3)
Pri1 (47.7)
Pol α (340.6)a
p180 (165.9)
p68 (66.0)
p55 (58.8)
p48 (49.9)
DNA polymerase/primasea
DNA polymerase
Structural subunit
Interacts tightly with p48
RNA primase catalytic subunit
MCMa
MCM (605.6)a
Mcm2 (98.8)
MCM (535)a
Mcm2 (91.5)
Putative 3'-5' replicative helicasea
Phosphorylated by Dbf4-dependent kinase
Mcm3 (107.5)
Mcm4 (105.0)
Mcm3 (91.0)
Mcm4 (96.6)
Ubiquitinated, acetylated
Helicase with MCM6,7; phosphorylated by CDK; aka Cdc54
Mcm5 (86.4)
Mcm6 (113.0)
Mcm7 (94.9)
Mcm5 (82.3)
Mcm6 (92.3)
Mcm7 (81.3)
Aka Cdc46; Bob1 is a mutant form
Helicase with MCM4,7
Helicase with MCM4,6; ubiquitinated
RPA (114)a
RPA (100.5)a
Single-stranded DNA-binding proteina
RPA70 (70.3)
RPA30 (29.9)
RPA70 (70.3)
RPA30 (29)
Binds DNA, stimulates Pol α
Binds RPA70 and 14, phosphorylated
RPA14 (13.8)
RPA14 (13.5)
Binds RPA30
PCNA
Pol δa
Pol εa
RPAa
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
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
Eukaryotic DNA Replication
z
z
Many conserved molecular
mechanisms
z Two separate DNA
polymerases δ/ε for the
leading strand and α for the
lagging strand
Differences
z Multiple Oris exist that fire in
a coordinated way
z Euchromatin replicates
first
z Larger, less well defined ori
sequences
z Old nucleosomes stay
attached and new
nucleosomes are added
z Preori complexes stable on
DNA through cell cycle –
activated by S-cdk