University of Groningen
A single-molecule view of DNA replicationGeertsema, Hylkje J.; van Oijen, Antonius; Chiu, Wah; Wagner, Gerhard
Published in:Current Opinion in Structural Biology
DOI:10.1016/j.sbi.2013.06.018
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Citation for published version (APA):Geertsema, H. J., van Oijen, A. M., Chiu, W. (Ed.), & Wagner, G. (Ed.) (2013). A single-molecule view ofDNA replication: the dynamic nature of multi-protein complexes revealed. Current Opinion in StructuralBiology, 23(5), 788-793. DOI: 10.1016/j.sbi.2013.06.018
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A single-molecule view of DNA replication: the dynamic nature ofmulti-protein complexes revealedHylkje J Geertsema and Antoine M van Oijen
Recent advances in the development of single-molecule
approaches have made it possible to study the dynamics of
biomolecular systems in great detail. More recently, such tools
have been applied to study the dynamic nature of large multi-
protein complexes that support multiple enzymatic activities. In
this review, we will discuss single-molecule studies of the
replisome, the protein complex responsible for the coordinated
replication of double-stranded DNA. In particular, we will focus
on new insights obtained into the dynamic nature of the
composition of the DNA-replication machinery and how the
dynamic replacement of components plays a role in the
regulation of the DNA-replication process.
Addresses
Zernike Institute for Advanced Materials, Centre for Synthetic Biology,
University of Groningen, 9747 AG Groningen, The Netherlands
Corresponding author: van Oijen, Antoine M ([email protected])
Current Opinion in Structural Biology 2013, 23:788–793
This review comes from a themed issue on Biophysical methods
Edited by Wah Chiu and Gerhard Wagner
For a complete overview see the Issue and the Editorial
Available online 24th July 2013
0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.sbi.2013.06.018
The power of single-molecule experiments lies in their
ability to reveal the dynamic behavior of individual
molecules and thereby obtain information on unsynchro-
nized, stochastic events that otherwise would be hidden
by ensemble averaging. In particular, single-molecule
tools greatly facilitate our ability to study complexes
consisting of multiple proteins by allowing the direct
observation of dynamics of association and dissociation
as well as the stoichiometry of protein components within
a complex. Besides providing quantitative information on
previously established molecular processes, the possib-
ility to follow in real time multi-protein complexes as they
undergo multiple sequential transitions has led to a
number of novel mechanistic insights [1�,2�,3�]. In this
review, we describe recent advances in single-molecule
studies of one such multi-protein complex, the DNA
replication machinery, and how these studies have
revealed dynamic properties that might be widely shared
with other multi-protein systems.
An intensively studied model system for DNA replication
is the bacteriophage T7 replisome, mainly because of
its relatively simple composition (Figure 1a) while
displaying a remarkable resemblance with more complex
replisomes of higher organisms [4,5]. Replication of DNA
is initiated by the unwinding of parental double-stranded
DNA (dsDNA) by the helicase domain of the T7 gene
protein 4 (gp4) [6,7], yielding two single-stranded DNA
(ssDNA) templates. The ssDNA is covered by single-
stranded binding proteins (gp2.5), which play a critical
role in coordinating the intermolecular interactions be-
tween the replication proteins. After unwinding, DNA
polymerases incorporate complementary nucleotides to
each of the ssDNA templates. The T7 DNA polymerases
consist each of a T7-encoded protein (gp5) and a pro-
cessivity factor thioredoxin (trx) [8], which is provided by
the host organism Escherichia coli. As is the case for all
DNA polymerases, they can only support DNA synthesis
in the 50–30 direction. Consequently, one of the strands,
the so-called leading strand, can be copied continuously
but the other strand, the lagging strand, is replicated in a
discontinuous fashion. To allow synthesis of the lagging
strand in a direction opposite to that of the leading strand,
the lagging strand is hypothesized to form loops that
permit the lagging-strand DNA polymerase to remain
associated with the rest of the replication complex [9].
The discontinuous replication of the lagging strand
results in the synthesis of short fragments, Okazaki frag-
ments, with a length of �1 kbp. Okazaki-fragment syn-
thesis is initiated from short RNA primers that are
synthesized by the primase domain of gp4.
Single-molecule experiments that relied on a real-time
readout of the length of a hydrodynamically stretched
DNA molecule (Figure 1b) resulted in the first direct,
real-time observation of the dynamic nature of enzymatic
processes that underlie DNA replication. For example,
such experiments demonstrated that primer synthesis
temporarily stalls the replisome to prevent leading-strand
synthesis from outpacing lagging-strand synthesis [10]
and visualized the formation and release of replication
loops during coordinated, leading-strand and lagging-
strand synthesis [11] (Figure 1b,c). Previous biochemical
studies suggested two possible mechanisms for replica-
tion loops to be released after the production of an
Okazaki fragment. The so-called collision model
describes the release of the polymerase from the lag-
ging-strand when it encounters the 50 end of the pre-
viously synthesized Okazaki fragment, whereas the
signaling mechanism will cause the replication loop to
be released when a new primer is synthesized, even
before the nascent Okazaki fragment is finished [11–13]. After finishing an Okazaki fragment, the lagging-
strand polymerase can be recycled to synthesize the next
Available online at www.sciencedirect.com
Current Opinion in Structural Biology 2013, 23:788–793 www.sciencedirect.com
Okazaki fragment or dissociates from the replisome, in
which case a new polymerase has to be recruited to
synthesize the next Okazaki fragment. The single-mol-
ecule observation of loop lengths and pauses between
loop-formation events allowed for a characterization of
these release mechanisms in the context of a fully active
replication complex. These studies resulted in a model
that the active replisome employs both collision and
signaling mechanisms likely as a fail-safe method to
ensure successful loop release.
With such a complicated choreography of proteins at the
replication fork it is important to understand how the
composition of the complex evolves over time. Numer-
ous biochemical and biophysical studies have contribu-
ted tremendously to the detailed knowledge of the
structure of replication proteins and the interactions
between them (reviewed in [4,5,14]), but a clear un-
derstanding of how these individual proteins interact
dynamically is still largely lacking. Recent single-mol-
ecule experiments have begun to suggest a picture of a
highly dynamic replisome that is characterized by a
continuous exchange of components [15��,16]. These
observations are emphasizing that the replisome does
not have the static composition often suggested by the
textbook pictures, but instead that it is a continuously
changing complex, dynamically exchanging components
while replicating DNA. In this review, we will focus on
the highly dynamic exchange of polymerases at the
replication fork, and argue that similar exchange mech-
anisms may be important to support subunit exchange in
many other processes.
The precise nature of the molecular interactions be-
tween the DNA polymerases and the rest of the repli-
some is one of the important determinants in processes
such as loop release and polymerase recycling. Bio-
chemical studies revealed that the T7 DNA polymerases
are physically tethered to the T7 gp4 helicase by two
different binding interactions [17,18]. During DNA syn-
thesis, an interaction between the helicase and the palm
domain of the polymerase keeps the polymerase tethered
to the replisome with high affinity. Alternatively, a
weaker, electrostatic interaction can be formed between
the acidic and unstructured C-terminal tail of gp4 and a
basic patch on the outside of the T7 polymerase. The
A single-molecule view of DNA replication Geertsema and van Oijen 789
Figure 1
Leadingstrand-derived
Laggingstrand-derived
Looprelease
DN
A shortening (μm
)
0
Loop
leng
th (
kb)
0.3
0.6
0.9
1.2
1.0
0
BE
AD
MO
VE
ME
NT
2.0
3.0
4.0
0 50 100 150 200 Time (s)
Laggingstrand
Leadingstrand
Previous OF
(a)
(b) (c)
gp5RNA primer
Nascent OF
ssDNAtemplateof next OF
gp4Helicase
Zinc-binding motifRNA polymerase
trx5′
5′ gp2.5
Current Opinion in Structural Biology
(a) Schematic view of the T7 bacteriophage replication machinery. (b) Experimental design to visualize replication-loop formation (blue arrow) and
release (red arrow) by the length changes of hydrodynamically stretched DNA molecules. (c) Length changes over time of a single DNA molecule
during replication. Loop formation and release are visible as a transient shortening of the flow-stretched DNA.
(a) Adapted from [4]; (b,c) adapted from [11].
www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:788–793
latter interaction does not require a primer-template for
the polymerase to be present. The availability of two
interactions with different binding stability is hypothes-
ized to mediate a switching mechanism that provides on
the one hand stable binding of polymerases while repli-
cating and, on the other hand, allows the polymerase to be
tethered loosely to the replisome. Earlier biochemical
experiments showed that even though T7 replisomes are
highly resistant to dilution [19], leading-strand and lag-
ging-strand polymerases showed rapid exchange when
challenged with excess polymerase in solution [20]. On
the basis of these experiments and similar ones on the T4
replisome [21], a switching mechanism was hypothesized
that would both confer high processivity to the polymer-
ase via a stable conformation supporting synthesis and
allow polymerase exchange via a more loose secondary
interaction with the replisome.
The dynamic nature of these interactions between the
polymerases and the helicase in the T7 system raises an
immediate paradox. How can the replisome processively
replicate thousands of basepairs, while polymerases are
able to readily exchange? A recent study by Loparo et al.[22��] utilized a novel single-molecule approach to
address this question. By combining the observation of
the DNA replication rate of a single T7 replisome with
the visualization of the recruitment of individual fluor-
escently labeled DNA polymerases from solution to the
complex, the authors showed that DNA polymerases bind
to the helicase tens of seconds before they are utilized in
actual DNA synthesis. The authors showed that a poly-
merase is initially recruited via the helicase acidic C-
terminal tail, followed by an exchange of the synthesizing
polymerase by the newly recruited one. This exchange
event is initiated by a transient release of the DNA
synthesizing polymerase from the DNA template, pro-
viding all polymerases bound to the helicase an oppor-
tunity to compete for binding to the exposed primer-
template substrate. This two-step mechanism potentially
allows for up to six polymerases, associated with the six
acidic helicase C-terminal tails, to be readily available to
replace the DNA-synthesizing polymerase. In the
absence of polymerases in solution, and thus in the
absence of additional competing polymerases bound to
the helicase C-termini, the synthesizing polymerase
while still tethered to the helicase will simply rebind
the primer very rapidly after dissociation. Thus, the
replisome ensures high processivity even in the absence
of excess polymerase.
Over the past years, both recruitment of more poly-
merases to the replisome than minimally necessary for
coordinated DNA replication and rapid exchange of
polymerases have been observed in other organisms,
suggesting that the aforementioned exchange mechanism
may be a general one. First, the leading-strand and
lagging-strand polymerases of the bacteriophage T4 have
been shown to form a homodimer [23], but both poly-
merases exchange within a minute when they are chal-
lenged by a mutant polymerase [21]. The polymerase
exchange mechanism is found to be induced by the
attachment of an extra polymerase to the clamp, which
competes for DNA binding with the replicating polymer-
ase. In addition, the T4 replisome has also been observed
to be able to bind up to three polymerases [24]. These
observations suggest that polymerase exchange within
the T4 replisome may follow a similar exchange mech-
anism as has been reported for the T7 replisome. Further-
more, polymerase exchange has also been observed for
the bacterial replication machinery of E. coli, both in vivoand in vitro. In E. coli, polymerases are tethered to the
replisome by the t subunits of the clamp loader, which in
turn connect to the helicase (DnaB) [25,26]. A clamp
loader can contain up to three t subunits, each of which
can bind a polymerase during DNA replication [27].
Notably, recent single-molecule in vivo experiments have
confirmed the biological relevance of having three poly-
merases at the replisome [28] and of frequent and
dynamic polymerase exchange within the replisome in
living E. coli cells [29��].
The biological advantage of the presence of a ‘backup’
polymerase may be most apparent when the replisome
encounters a lesion in either the leading-strand or lag-
ging-strand template, when the lagging-strand polymer-
ase is left behind due to premature stalling of Okazaki-
fragment synthesis, or generally when one of the poly-
merases loses functionality. The E. coli replication
machinery overcomes lesions in the DNA template by
either bypassing the lesion by generating a downstream
primer on the leading strand from which processive DNA
replication can restart [30] or repairing the lesion by
rapidly exchanging the high-fidelity polymerase III for
the low-fidelity lesion bypass polymerase IV. The rapid
polymerase exchange is regulated by the b sliding clamp,
which can bind both polymerases simultaneously [31].
The presence of an extra polymerase at the replication
fork may provide the opportunity to quickly load a poly-
merase on the downstream primer to continue leading-
strand synthesis as well as the possibility to exchange a
processive polymerase by an error-prone polymerase to fix
lesions. Additionally, single-molecule measurements
demonstrate that replisomes containing three poly-
merases are more efficient in nucleotide incorporation
and generate shorter ssDNA gaps in the lagging strand
than dual-polymerase replisomes [32�]. Dohrmann et al.[33] recently showed that the dissociation rate of a single
polymerase from the 30 end of a DNA template is too slow
to successfully recycle lagging-strand polymerases.
Taken together, these studies strongly suggest a picture
in which the third polymerase binds a newly formed
primer to start synthesis of the next Okazaki fragment,
such that the former lagging-strand polymerase can finish
the Okazaki fragment, possibly by being released from
790 Biophysical methods
Current Opinion in Structural Biology 2013, 23:788–793 www.sciencedirect.com
the replisome, or remains bound long enough to be
recycled to the second-next Okazaki fragment.
In this review, we have focused on the molecular mech-
anisms underlying stable binding and high processivity
of polymerases at the replisome while allowing rapid
exchange. Interestingly, recent single-molecule exper-
iments have revealed similar dynamic subunit exchange
mechanisms in other multi-protein complexes. FRAP
(fluorescence recovery after photobleaching) and FLIP
(fluorescence loss in photobleaching) experiments demon-
strated that the MotB and FliM subunits of the bacterial
flagellar motor are exchanged in a signal-dependent way
[34,35]. In addition, the structural maintenance of chromo-
some proteins of E. coli, MukBEF, was determined to form
multimers that can exchange single MukBEF complexes
with freely diffusing complexes in the cytosol [36,37].
The multimeric form of MukBEF is proposed to allow
the release of one DNA segment without releasing
the whole multimeric complex from the chromosome.
The observation of multiple examples of stable multi-
protein complexes that can readily exchange subunits
suggests a more general picture of multi-protein complex
that are able to dynamically exchange components with
spare parts available in solution while maintaining high
stability in absence thereof.
We hypothesize that the dynamic exchange of subunits
within multi-protein complexes while maintaining its
overall composition and structure is a widespread mech-
anism. Stable binding of subunits while allowing rapid
exchange is made possible by the presence of multiple
sites of interaction between the subunit and the rest of
the complex. Such a stable binding would be mediated by
binding a subunit at two sites simultaneously. A transient
disruption of one of the two interactions would not
immediately result in dissociation of this subunit but
would allow, however, a second subunit from solution
to bind at the vacated binding site. The presence of two
subunits would then be quickly resolved by one of them
competitively displacing the other altogether. Such a
mechanism could be compared to two monkeys reaching
for the same hand of bananas while swinging from a tree
branch. A monkey holding the branch can stably grab the
bananas, but a transient release will allow another
monkey, associated with a second binding site, to join
in and potentially take over the desired bananas (Figure 2,
left). However, the ability to stably hold on to the branch
allows the monkey to keep its position, close to the
bananas. In the absence of a competing monkey,
the secondary binding site allows the monkey to quickly
take hold again of the bananas after having released them
(Figure 2, right). Single-molecule techniques are very
well suited to characterize such exchange mechanisms
at the molecular scale. The technical advances in these
methodologies and our increasing understanding of the
molecular details of the interactions involved place us in
an ideal situation to further study and understand such
A single-molecule view of DNA replication Geertsema and van Oijen 791
Figure 2
Current Opinion in Structural Biology
Schematic representation of entities competing for a single preferential binding site. (left) Competition of two monkeys for the same preferential binding
site (a hand of bananas). Transient dissociation from the bananas by the monkey on the right allows the left monkey to compete for the same bananas.
(right) With just one monkey present, temporary unbinding from the bananas still allows a rapid re-association by the same monkey.
www.sciencedirect.com Current Opinion in Structural Biology 2013, 23:788–793
molecular gymnastics with unprecedented dynamic
detail.
AcknowledgementsAMvO would like to acknowledge funding from the NetherlandsOrganization for Scientific Research (NWO; Vici 680-47-607) and theEuropean Research Council (ERC Starting 281098).
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