University of Groningen

A single-molecule view of DNA replicationGeertsema, Hylkje J.; van Oijen, Antonius; Chiu, Wah; Wagner, Gerhard

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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 (a.m.van.oijen@rug.nl)

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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� of special interest

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