Assembly dynamics of supramolecular protein-DNA complexes studied by single-molecule
fluorescence microscopy
Stratmann, Sarah
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supramolecular protein-DNA
complexes studied by single-
molecule fluorescence microscopy
ingen, The Netherlands. The research was financially supported by the Netherlands Organ-isation for Scientific Research (NWO), European Research Council (ERC) and the Zernike Institute for Advanced Materials.
Copyright © 2017 Sarah Stratmann
All rights reserved. No part of this publication may be produced, stored in a retrieval system of any nature, or transmitted in any form or by any means, electronic, mechanical, including photocopying and recording, without prior written permission of the author.
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Cover design and layout: Sarah Stratmann & Bastian Niebel ISBN: 978-90-367-9496-1 (printed version)
DNA complexes studied by single-molecule
fluorescence microscopy
Proefschrift
ter verkrijging van de graad van doctor aan de
Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
maandag 20 februari 2017 om 09.00 uur
door
Sarah Annette Stratmann
geboren op 6 april 1986
Prof. dr. A.M. van Oijen
Beoordelingscommissie
Prof. dr. G. Maglia
Prof. dr. S. Diez
Chapter 1: Introduction 9
1.1 DNA metabolism 10 1.2 DNA replication 10 1.3 DNA sensing by the auto-immune system 12 1.4 Aim of this thesis 14 1.5 References 15
Chapter 2: DNA replication at the single-molecule level 19
2.1 Introduction 20 2.2 Experimental strategies to image single molecules 22 2.2.1 Getting proteins to shine 22 2.2.2 Trapping and pulling at individual DNA molecules 27 2.3 Replication machineries 28 2.3.1 Model systems for single-molecule studies 30 2.3.2 Replication-fork assembly pathways 34 2.3.3 Leading and lagging-strand coordination 36 2.3.4 Polymerase dynamics 38 2.3.5 In vivo studies on the E. coli replisome 40 2.3.6 Replication in the context of eukaryotic cell division 44 2.4 Conclusions and Outlook 48 2.5 References 48
Chapter 3: Single-molecule studies of DnaB loading and dynamics at the Escherichia coli
replication fork 59
3.1 Introduction 60
3.2 Results 62
3.2.1 DnaB – association kinetics at forked DNA 62 3.2.2 Loading of multiple helicases at the replication fork 64 3.2.3 DnaB is stably integrated into the replisome during unwinding 65
3.6 References 74
Chapter 4: The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by
scanning along the duplex 77
4.1 Introduction 78 4.2 Results and Discussion 78 4.3 Materials and Methods 84 4.4 Supplementary data 88 4.5 References 92
Chapter 5: Bisecting microfluidic channels with metallic nanowires fabricated by nano-skiving: Applications in flow sensing and single-molecule fluorescence studies 95
5.1 Introduction 96 5.2 Results and Discussion 97 5.2.1 Fabrication 97 5.2.2 Hot-wire anemometry 99 5.2.3 Suspended DNA curtains 101 5.2.4 Single-molecule studies on suspended DNA curtains 104 5.3 Conclusion 106 5.4 Materials and Methods 107 5.6 References 113
Chapter 6: Summary - Samenvatting 115
Summary and future perspectives 116 Samenvatting en toekomstperspectief 118
References 121
Introduction
1.1 DNA metabolism
“14% N, 5.8% P, 1.8% S.” This result of the chemical analysis of purified nuclei from
leu-cocytes by Friedrich Miescher in 1871 (1) heralded a new era of research in physiological chemistry. It let the author speculate about a so far unknown chemical moiety that he named ‘nuclein’, richer in phosphor and nitrogen than any other known protein. As it turned out, Miescher succeeded for the first time to extract DNA from cell nuclei. Following Miescher’s work, Albrecht Kossel decoded the chemical groups of nucleic acid, the five bases adenine, thymine, guanine, cytosine, and uracil. Remarkably, Kossel realized that proteinaceous mass was co-purified with the nucleic acids from nuclei, indicating a more complex and perhaps biologically relevant structure. He suggested a novel class of nucleic-acid associating proteins that he named histones (2). He postulated that these basic proteins, rich in lysine and argin-ine, might have transformed from ordinary proteins to interact with nucleic acids in ‘chromi-oles’ (later called chromosomes) through ionic interactions (3).
Today we know that DNA-interacting proteins play roles in every aspect of DNA metabolism: A machinery of enzymes replicates DNA, specialized enzymes constantly repair errors in the bases to prevent genomic mutations, structural proteins package DNA into three-dimen-sional structures, and nucleases degrade DNA in the process of cell death. Besides all these enzymes involved in processing DNA, a large number of proteins and enzymes are involved in transcription and translation and the vast regulatory network associated with it.
This thesis deals with two topics in DNA metabolism, investigated using biochemical as well as single-molecule approaches: Assembly of DNA-replication complexes in E. coli, and the sensing of pathogenic DNA in mammalian systems. These two processes represent illustrative examples of the maintenance and processing of genomic material and the regulation required to control these processes. DNA replication is a highly regulated process in all organisms. In prokaryotes, nutrient supply represents a major factor triggering replication, whereas in eukaryotes, cell-cycle factors determine whether the cell-division phase is to be entered. The identification of pathogenic DNA also presents a tightly regulated mechanism in the context of propagation of genomic material, albeit a negative one. Eukaryotic cells have developed specific sensors that detect DNA and other forms of nucleic acid belonging to invading patho-gens and that prevent their replication in the context of an inflammatory response. In the following sections I will provide a brief background summary on both topics and define the various research questions as a context for the work I present in this thesis.
1.2 DNA replication
Organisms have developed complex strategies to regulate the timing of replication initiation, to ensure that the replication of genomic material is timed correctly in the context of the cell-division cycle. In eukaryotes, multiple enzymes are involved in initiating the formation of a large number of replication complexes on various positions on the chromosomal DNA. The key proteins in this process are subject to a variety of cell-cycle dependent activation processes, such as phosphorylation by cyclin-dependent kinases leading to protein
ubiquiti-nation and degradation or export from the nucleus (4). In prokaryotes, regulatory systems of replication initiation have been shown to involve fewer regulatory components and are regu-lated more by the environment than an internal cell cycle. In rich media, for example, cells can grow with overlapping replication cycles, enabling cell-division times shorter than one round of genome replication. Here, rigid and global regulation of initiation allows simultaneous firing on partially replicated chromosomes (5).
Replication of the circular E. coli chromosome is initiated at a single origin of replication, a 245-bp specific sequence called oriC. Key regulator of DNA replication is the AAA+-family ATPase DnaA that binds to the five 9-bp long DnaA boxes within oriC. Oligomerization of ATP-bound DnaA along DNA induces positive supercoiling and local melting of the three nearby located AT-rich 13-bp long sequences called DUE (DNA unwinding element). This local unwinding then allows binding of two complexes that each consist of the replicative helicase DnaB and its loader protein DnaC (6-9). The next steps of replication depend on the concerted action of several key players at the replication fork. The ring-structured rep-licative helicase DnaB encircles single-stranded DNA and couples the energy released from nucleotide hydrolysis to directional movement und unwinding of the parental duplex. DNA polymerase III holoenzyme, a complex of eight different subunit proteins, synthesises the two daughter DNA strands, starting from RNA primers produced by the primase DnaG (a detailed description of the bacterial replisome is given in Chapter 2).
Intriguingly, these replisomal machineries cannot be purified as intact complexes - in con-trast to for example the macromolecular ribosome (10). The paradoxal situation of a complex that supports highly processive replication of millions of basepairs while being held together with weak interaction has been subject of recent research studies (11-13). These studies have shown that multi-site interactions within the replication complex seem to allow dynamic ex-change of DNA polymerases and polymerase holoenzymes while ensuring robustness of the complex. With the classical picture of a stable replisome recently being challenged, the ques-tion arises whether the replicative helicase DnaB remains stably integrated in the replisome (14, 15) or whether it also dynamically exchanges protein components with those present in
Figure 1: DNA replication in E. coli. A) At the oriC sequence, two replication complexes are established that progress
bidirectionally along the chromosome, until they are stopped at the termination (Ter) sites. B) The initiator protein DnaA oligomerizes at the oriC, unwinding the AT-rich domains. Two complexes that each contain a DnaB helicase and DnaC helicase loader assemble onto the single-stranded region and trigger the formation of a pair of replisomes.
oriC Ter sites oriC Ter sites Bidirectional fork progression oriC AT domains DnaA boxes DnaA HU DnaB DnaC Pre-priming complex IHF A B
solution. In this thesis, we challenged the stability of DnaB during loading and replication fork progression.
1.3 DNA sensing by the auto-immune system
Regulation of DNA replication initiation not only impacts the cellular division cycle, but also presents an important factor in combating invading pathogens and preventing parasitic rep-lication. This process lies at the interface of DNA metabolism and cellular immunity. In both bacteria and eukaryotes, immune systems have been discovered that either act adaptively against invading pathogens and build a immunological memory against the respective or-ganism, or that are encoded in the innate immune system, providing a direct and broader response.
In bacteria, the CRISPR/Cas system has been recently identified to serve as an efficient adap-tive immune strategy that memorizes past invasions of bacteriophages (16, 17). An innate defense mechanism is provided by the restriction-modification system, relying on the ex-pression of restriction enzymes that cleave specific sequences of non-methylated DNA (18). To protect the self-DNA, DNA methylase enzymes methylate the corresponding sequences within the genome immediately after replication.
Vertebrates possess highly complex adaptive and innate immune systems to respond to pathogenic threats. Whereas adaptive immunity is based on clonal gene rearrangements to express antigen-specific receptors, the innate immune system has been recognized as more general (19). Innate immune reactions present the first line of defense, with their activation upon pathogenic infection and cell invasion, and seem to be a prerequisite for activation of the adaptive immune system (19-21). Several classes of sensors, named Pattern-Recognition Receptors (PRRs), have been identified that detect structural elements of pathogens, so-called Pathogen-Associated Molecular Patterns (PAMPs), and that trigger signaling cascades for the expression of pro-inflammatory molecules. Usually, PAMPs are structural elements such as nucleic acids, surface glycoproteins, lipoproteins and membrane components of pathogens, each recognized by specialized PRRs (19). Pathogenic membrane structures such as glyco-proteins are recognized by the immune system as foreign, since these structures normally do not exist inside the eukaryotic host cell. But how does a cell distinguish foreign DNA from its own genomic material? Different scenarios are possible: 1) an exclusively cytoplasmic lo-calization of the PRR, 2) detection of specific motifs of pathogenic or damaged DNA such as unmethylated CpG islands, or 3) detection of structural properties of foreign DNA such as the lack of tight nucleosomal packing (20, 22). The strict discrimination between foreign and self-DNA is a substantial requirement for the proper activation of inflammatory reactions. As a consequence, auto-immune disorders often result from misregulation in the process of foreign-DNA detection or downstream processes (23, 24).
A number of DNA-recognizing PRRs have been reported to be confined to the cytoplasm, like AIM-2 (25), or to exclusively detect pathogen-specific DNA motifs such as CpG islands, like TLR-9 (26, 27). Amongst the different human PRRs that detect DNA, the IFI16 protein is unusual in that it is present within the cytoplasm as well as the nuclear region (28-30).
Figure 2: Pathogen detection by PAMP sensing. Pattern recognition receptors such as Toll-Like-Receptors (TLR’s),
AIM2, and IFI16 have as a key task the detection of certain foreign structures. IFI16 is present in the nucleus, where it senses pathogen DNA and triggers export of the DNA to the cytoplasm. STING and ASC/caspase-1 are assumed to interact with the IFI16-DNA complex and start the inflammatory response.
First shown to be engaged in a p53-mediated apoptotic pathway (31, 32), IFI16 is now mostly thought to be involved in immune mechanisms against invading pathogens, such as Kaposi’s sarcoma-related herpes virus (KSHV), HIV, listeria or salmonella (28, 33-35). IFI16 belongs to the interferon (IFN)-inducible p200-protein family, with two 200-amino acid long do-mains (HIN-A and HIN-B) at the C-terminus that interact sequence-independently with the sugar-backbone of the DNA (36), and an N-terminal Pyrin domain. It has been demonstrated that the tandem-arranged HIN domains each bind independently to dsDNA, whereas the Py-rin domain promotes protein-protein interactions (37). An N-terminal nuclear localization sequence enables transport to the nucleus, with lysine acetylations playing a regulatory role in the cellular localization (38).
Upon foreign-DNA detection, IFI16 activates an inflammatory response and restricts the rep-lication and transcription of pathogenic DNA in infected cells. Orzalli and co-workers have shown that IFI16 mainly limits un- or underchromatinized viral DNA, when comparing the effects on transfected bare SV40 DNA with those seen in SV40 viral infection, with the viral DNA nucleosomally packaged by host-cell histones (39). After detection of pathogenic DNA, the IFI16-DNA complex is exported to the cytosol and activates STING (stimulator of inter-feron genes) (40) and caspase-1 (41). Regulation mechanisms of IFI16-induced inflammatory responses are not understood in depth, however a cellular antagonist, the Pyrin-domain only protein (POP3), was shown to inhibit IFI16-mediated processes (42, 43). Together with in
vitro analyses that proved that the Pyd domain is responsible for oligomerization, catalyzed
by binding to DNA (37), inflammatory IFI16 action is assumed to rely on very specific aggre-gation on foreign DNA. How this detection and aggreaggre-gation process is achieved, has been one of our main interests in this thesis.
Nucleus Cytoplasm Parasites Viruses Pathogen Associated Molecular Patterns Sensors STAT6STAT6chemokines NF-κB inflammatory responses ER STING ASC Casp AIM2 TLR9 IFI16 PolIII Rig-1 cGAS IFI16 IFI16
1.4 Aim of this thesis
As seen from this brief overview, DNA replication, sensing, and control are key biological processes. In this thesis, using state-of-the-art fluorescence microscopy techniques and data analysis tools, we aim at unraveling main aspects of DNA metabolism on the single-molecule level. Real-time, non-invasive techniques such as single-molecule fluorescence imaging offer insights into transient biochemical steps in DNA interaction or enzymatic cycles and help us identify intermediates in DNA-protein complex assembly. We profit from classical biochemi-cal techniques that enable the high-throughout generation of protein-DNA interaction maps (44, 45) and elucidate structural motifs of DNA sequence specificity by proteins by atomic resolution structures (46, 47), and bulk activity assays to resolve DNA metabolism by special-ized enzymes. By observing at the single-molecule level assembly steps of the IFI16 inflam-matory complex on DNA, as well as of the replicative DnaB helicase within the replisomal complex, we can quantify binding stabilities of single molecules versus protein complexes, characterize DNA-binding modes and search mechanisms along DNA.
In Chapter 2, we summarize biophysical developments that have contributed to our under-standing of the fundamental mechanisms underlying DNA replication. We focus on technol-ogies especially in fluorescence microscopy and their applications in single-molecule studies on different replication systems such as bacteriophage T7, E. coli and eukaryotic cells. We strengthen the impact of high-resolution techniques to build mechanistic models of multi-enzymatic and multiprotein reactions en detail, providing spatial and time information of intermediate reactions and protein interactions that cannot be achieved by ensemble averag-ing methods.
In Chapter 3, we implement single-molecule tools to characterize the dynamics of the bac-terial helicase DnaB at the replication fork. We show that DnaB stably associates at artificial DNA forks and allows subsequent replisome assembly, thereby supporting efficiently replica-tion initiareplica-tion. Relative to the lifetimes of all other replisomal components and the disconti-nuity frequency during coupled replication, we show that DnaB remains stably incorporated within the replisome and thus may act as the central organizing hub within the replisome to provide its overall integrity.
In Chapter 4, we use a combined biochemical and biophysical approach to unravel the nu-cleic-acid detection mechanism of the human DNA sensor IFI16. We show that IFI16 scans along duplex DNA with a high one-dimensional diffusional velocity to search for other IFI16 molecules, eventually forming a multi-protein complex stably associated with DNA. This complex serves as signaling platform to trigger inflammatory pathways and is physiologically switched on upon pathogen invasion into the cellular nucleus. We show that highly specific sensing of foreign DNA relies on the increased reaction cross section that IFI16 can exploit on under-chromatinized genomes.
Chapter 5 comprises a methodological advance related to practical challenges encountered in observing DNA-protein interactions at the single-molecule level. One major hurdle in the research described in this thesis is the passivation of support surfaces to prevent nonspecific interactions between these surfaces and the protein-DNA complexes under study. In Chap-ter 5, we describe the development of a novel microfluidic chip design based on suspended
nanowires. We span gold nanowires across channels and apply a straightforward protocol for functionalization and biomolecule attachment. This configuration allows us to analyze protein-DNA associations and dynamics even at high protein number per DNA molecule. We use IFI16 diffusion and aggregation along DNA to demonstrate the advantages of the suspended nanowire configuration compared to established surface immobilization protocols in single molecule microscopy.
1.5 References
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DNA replication at the single-molecule level
Sarah Stratmann and Antoine van OijenChem Soc Rev. 2014 Feb 21;43(4):1201-20
A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA repli-cation, RNA/protein synthesis or intracellular transport. By purifying single components and using them to reconstitute molecular processes in a test tube, researchers have gath-ered crucial knowledge about mechanistic, dynamic and structural properties of biochemi-cal pathways. However, in order to sort this information into an accurate cellular road map, we need to understand reactions in their relevant context within the cellular hierarchy, which is the individual molecule within a crowded, cellular environment. Reactions occur in a stochastic fashion, have short-lived and not necessarily well-defined intermediates, and dynamically form functional entities. With the use of single-molecule techniques these steps can be followed and detailed kinetic information that otherwise would be hidden in ensemble averaging can be obtained. One of the first complex cellular tasks that has been studied at the single-molecule level is the replication of DNA. The replisome, the multi-pro-tein machinery responsible for copying DNA, is built from a large number of promulti-pro-teins that function together in such an intricate and efficient fashion that make the complex robust to DNA damage, roadblocks or fluctuations in subunit concentration. In this review, we sum-marize advances in single-molecule studies, both in vitro and in vivo, that have contributed to our current knowledge of the mechanistic principles underlying DNA replication.
2.1 Introduction
Life is as dynamic as its environment. Many key cellular processes cannot be described as outcomes from static associations of molecular components, but instead rely on an intricate spatial and temporal orchestration of many molecular players. For example, the conversion of chemical energy into mechanical work allows the transport of vesicles and molecules within the cytosol, along a membrane or between cells. On the single-molecule level, kinesins and other motor proteins move along the cytoskeletal filaments, transporter proteins shuffle me-tabolites between compartments, and multi-subunit complexes like replisomes, ribosomes, or the respiratory chain support an efficient maintenance and balancing of anabolism and catabolism.
Both fluorescence- and force-based single-molecule studies have provided fascinating new insights into some of these elaborate biological processes, such as cytoskeletal dynamics (1-3), ATP synthesis (4, 5), RNA and DNA polymerization (6-8), and viral packaging motors (9, 10) . The more recent developments in life-cell single-molecule imaging allows us to record the cellular micro-management in real time, as has been demonstrated for example for tran-scription-factor dynamics (11), protein-expression rates (12), and signalling pathways (13) (reviewed in Ref. (14)).
What type of knowledge do we obtain from experiments monitoring individual molecules? Ensemble-averaging bulk assays provide information about the reaction rates of a pool of cat-alysts and, by synchronizing reactions, kinetic studies can reveal the first few transitions of a multi-step process. However, loss of synchronization due to the stochastic nature of chemical reactions will render it challenging to obtain kinetic parameters of short-lived intermediate states. Single-molecule studies capture the probabilities of reaction steps or conformational changes of an individual enzyme during any arbitrary point along a multi-step process and provide information on underlying heterogeneities in the dynamic behaviour of the popula-tion (15, 16). Watching individual reacpopula-tions at work tells us not only about the stochasticity of consecutive pathways, but also informs us about any temporal correlation: Does an enzymatic reaction for example display non-markovian behaviour, i.e. are reaction steps affected by pre-ceding steps (17)? One of the earliest single-molecule fluorescence studies demonstrated such a memory effect in a flavoenzyme: autocorrelations of on and off dwell times of the redox cofactor FAD(H2) resolved heterogeneous kinetic rates, caused by conformational changes
within the protein, that had been previously masked in bulk experiments (16). Similarly, sin-gle-molecule analyses of the RecBCD helicase of Escherichia coli could decipher subpopu-lations or microstates of the enzymatic complex that differ in the velocity of DNA unwind-ing(18). Here, conformational changes that are adopted in the absence of the ligand/substrate ATP are “memorized” by the active RecBCD upon ATP addition and result in distinct rates of progression along the DNA template.
The actual chemical conversions in enzymatic reactions typically proceed on a sub-picosecond time scale. However, the limiting steps in catalysis are often the crossing of thermal activation barriers or the diffusive process necessary to mediate association of two reactants, which last orders of magnitude longer and are consequently the parameter to follow in single-molecule studies (19, 20). Typical fluorescence assays rely for example on visualizing a chromophore coupled to a molecule of interest and monitoring the appearance and disappearance of its
signal as the labelled component is binding to and dissociating from a reaction partner mol-ecule (Figure 1). Binding life times can be extracted and a probability distribution generated that contains the kinetic rates of the observed reaction. Several reviews on single-molecule enzymology provide excellent descriptions of enzymatic kinetics based on single-molecule reaction probabilities (16, 21).
In addition to successes in resolving single-protein kinetics, recent developments have fo-cused on the visualization of protein dynamics and complex assemblies in real time, usually using fluorescence co-localization or fluorescence (Förster) resonance energy transfer (FRET) methods. These approaches have allowed, for example, the observation of the dimerization of EGF receptors in living cells, the complex formation of a reconstituted functional vesicle fusion construct of t- and v-SNARE proteins, or the Arp2/3-mediated branch formation on growing actin filaments (13, 22, 23). Studies of the replisome, the machinery responsible for DNA replication, face the challenge of revealing the various and frequently transient interac-tions of the numerous enzymes that are involved (24-26). The multi-component replisome is loaded on the DNA template in tight coordination with the cell cycle, it proceeds with a speed of up to thousand nucleotides per second (for certain bacterial systems), corrects wrongly incorporated nucleotides to an accuracy of about one mistake per 109 nt and triggers repair
processes upon detection of depurination, deamination, or pyrimidine-dimer formation (27, 28). Coordination of such a wide array of tasks, each on their own representing formida-ble molecular challenges, requires a finely tuned and balanced set of enzymatic activities. Building on the large base of knowledge we have on the individual components of the rep-lication reaction, derived from many decades of genetic, biochemical and structural studies,
Time
Intensity/Photon counts
Pr
obability
Dwell time of “on“ state
A
B
Fitting “on“
“off“
Figure 1: Extraction of single-molecule kinetics
from the observation of on and off times. These on- and off times can represent a variety of func-tional or structural transitions such as binding/ unbinding, conformational transitions or chemi-cal reactions. A) On- and off times of an observed fluorescent emitter are recorded and the photon count per molecule is tracked over time and fitted to an appropriate function. B) The time scales for on and off times are sorted in a distribution that provides the kinetic parameters of the individual reaction.
single-molecule approaches represent a powerful approach to unravel the intricacies of how the various enzymatic activities at the replication fork are coordinated.
The process of replication needs to deal with a variety of molecular hurdles. For example, the antiparallel nature of the double-stranded DNA template imposes an asymmetry on the replication machinery, whose DNA polymerases can only synthesize in one particular direc-tion. Besides the need for this asymmetric coordination, other obstacles have to be tackled, such as crowding effects and roadblocks caused by transcription-related processes and repair activities that take place simultaneously on the same DNA template. How exactly cells meet those challenges is a subject particularly well-suited for single-molecule studies – requiring methods to observe the spatiotemporal behaviour of individual molecules in a biologically relevant environment.
In this review, we describe recent developments in single-molecule research on the replisome
in vitro and in vivo. We first dedicate a chapter on the main technological developments in
terms of microscope setups, design of fluorophores and labelling methods. Referring to the replication systems of the bacteriophages T7 and T4, of Escherichia coli (E. coli), and of eu-karyotic cells, we guide the reader through the different aspects of important single-molecule studies that have contributed to a better understanding of the basic mechanics of DNA repli-cation and organization.
2.2 Experimental strategies to image single molecules
Single-molecule techniques are typically categorized into two classes that we want to outline briefly: Fluorescence microscopy allows the recording of the emitted photons of a fluoro-phore-labelled molecule of interest and is particularly applicable for catching conformational changes within the protein of interest or its localization. Force-based measurements, like atomic force microscopy (AFM), magnetic tweezers, optical traps or flow-stretching setups, are useful in characterizing mechanical properties such as DNA topology or force exertion by motor proteins.
2.2.1 Getting proteins to shine
As early as in the 70’s, it was demonstrated that single protein molecules labelled with a large number of dyes could be detected in an optical microscope (29): Tomas Hirschfeld coupled roughly hundred fluorescein dyes to a single antibody, swept a dilute solution of these con-structs along a tightly focused laser beam, and observed bursts of fluorescence each cor-responding to a single antibody. Not until two decades later, absorption and fluorescence measurements of single chromophores were successfully performed at cryogenic tempera-tures where absorption cross-sections are highest and photo-induced damage lowest (30-32). Where initially these studies were performed on doped molecular crystals, later cryogenic single-molecule approaches were applied to study pigment-protein complexes (33). Near-field scanning microscopy approaches demonstrated the feasibility to repeatedly image chro-mophores within biological samples at ambient temperature (34), but were later joined by
even more powerful and technically less-demanding far-field methods, mainly confocal and total-internal-reflection (TIRF) microscopy. Since then, great advances in high-sensitivity detection devices, in engineering of photostable dyes and fluorescent proteins, and labelling strategies pushed the sensitivity and resolution limits to a point where single molecules can be observed over timescales from milliseconds to minutes and down to spatial resolutions of a few nanometres. Furthermore, advances in live-cell imaging have enabled such experiments in a cellular context. Here, additional factors have to be considered in terms of cell viability (nutrients, CO2, photodamage due to decomposition of fluorophores and radical release), and
fluorophore choice (uptake, label selectivity and specificity). Even though these developments are relatively recent and many novel methods are still coming to fruition, single-molecule approaches are already revolutionizing the way mechanistic questions of biological systems are answered.
Hardware technology
The optical instrumentation required for single-molecule imaging and tracking can be roughly divided in two modes of operation: wide-field imaging and scanning confocal mi-croscopy (Figure 2). Both approaches have their advantages and need to be adapted to the actual question in consideration of both spatial and temporal resolution.
Wide-field imaging is a frequently used method to follow reactions at the single-molecule level in real time, i.e. to track particles and observe fast dynamics. In epifluorescence micros-copy, a large sample volume is excited, limiting the signal-to-noise ratio in the region-of-in-terest. However, thin samples, either reconstituted isolated compounds or flat cells, can be analysed with single-molecule sensitivity, as shown for microtubule gliding on kinesins or live-cell protein expression (35, 36). Being proposed already in the 1950’s but not fully de-veloped until several decades later (37, 38), TIRF microscopy has proven to be exceptionally useful in improving signal detection. Here, at the coverglass/solution interface an evanescent field is induced that decays exponentially in the z plane and limits the excited volume to about 100 nm.
In confocal imaging, a diffraction-limited focus is positioned within the sample volume and scanned orthogonally to the optical axis (39). The use of pinholes results in the selective de-tection of only in-focus fluorescence, while suppressing most out-of-focus background. In
Sample Coverglass
Total int. reflection Focal plane
Epi-fluorescence Confocal Spinning disk
Figure 2: Fluorescence microscopy designs frequently used in single-molecule studies. In epifluorescence
micros-copy, the light source illuminates the entire sample. In confocal microsmicros-copy, a pinhole is used to illuminate specifi-cally the focal plane, thus reducing background fluorescence. By installing a Nipkow spinning disk, the sample is il-luminated at multiple points within the focal plane simultaneously. In Total-Internal-Reflection Fluorescence (TIRF) microscopy, the incident laser is reflected from the coverglass surface, creating an exponentially decaying evanescent field on top of the surface, that reduces the thickness of the illuminated volume to about 100 nm.
contrast to a TIRF setup, confocal microscopy allows the scanning of samples in three dimen-sions with large penetration depth. However, the limiting factor is the scanning speed of the focal spot through the sample. Spinning-disk confocal setups employ a broad laser illumina-tion that is focused in a large array of microlenses on a Nipkow disk, achieving high frame rates of up to 1000 frames per second (40).
Non-linear two-photon techniques use optical sectioning as well, but here focussing relies on the probability of two-photon absorption, which is proportional to the square of the excita-tion intensity. A main advantage is that the required lower-energy wavelengths reduce photo-damage of the fluorophores as well as scattering in tissue samples. Depths of several hundred micrometres are achievable with this method, as for example demonstrated in fascinating work on intact organs in living organisms (41).
Technological developments in optical microscopy have contributed to a gradual improve-ment of spatial resolution, but with the size of the smallest resolvable structures still similar to the diffraction limit. The recent breakthroughs in super-resolution imaging, however, have al-lowed the imaging of fluorescently labelled structures down to length scales that are an order of magnitude smaller than the diffraction limit. Super-resolution methods find their basis in the reduction of the point-spread function (PSF) in excitation, as in stimulated emission de-pletion (STED), ground-state dede-pletion (GSD) or structured illumination microscopy (SIM), or in the modulation of the fluorophore’s emission, as in photoactivated localization micros-copy (PALM) and stochastic optical reconstruction microsmicros-copy (STORM) (Figure 3). STED microscopy is based on the illumination of the sample with a doughnut-shaped beam profile. The excitation beam is narrowed by an overlaying ring-shaped longer-wavelength depletion beam, that forces the dyes into the ground state (42). The higher the intensity of the depletion
N cycles
S0
S1
Exc Depl Fluor
off on STED STORM/PALM T1 S0 S1 Exc Fluor on off/bleach
Figure 3: Super-resolution techniques. In STED, fluorophores are excited to the S1 state (green) and return to the
ground state S0 spontaneously while emitting photons (yellow). An intense red-shifted doughnut-shaped depletion
laser beam (red) forces molecules into the ground state without them emitting fluorescence. As a result, only a sub-diffraction-limited area in the center of the depletion laser remains in the excited state and will be observable through the emission of a yellow fluorescence photon. A similar excitation geometry is used in Ground State Deple-tion (GSD) microscopy. However, instead of rendering the fluorophores around a point of interest nonfluorescent by depleting the fluorescent excited state, they are brought into a long-lived dark state. In PALM and STORM, molecules are switched on at low spatial densities, their positions determined with sub-diffraction-limited precision, and irre-versibly photobleached. Repeating this procedure for a large number of molecules results in sub-diffraction-limited images (adapted from Ref. (191)).
laser, the narrower the PSF becomes. In a similar design, but typically using only one wave-length, GSD brings the fluorophores to their lowest triplet dark state in the outer ring. An alternative approach to super-resolution imaging is enabled by the wide-field methods PALM and STORM, utilizing the stochastic activation of fluorophores that are photoactivatable or photoswitchable. The activation of a few fluorophores in the field of view allows each of them to be individually imaged and to be fit by a two-dimensional point-spread function and thus each of their centroid positions to be obtained with sub-diffraction-limited precision. It is the sum of several cycles of activation - centroid detection - bleaching/inactivation that leads to the reconstruction of the complete object of interest. The super-resolution techniques PALM and STORM have also played important roles recently in resolving intracellular dynamic pro-cesses at the single-molecule level (e.g. (43, 44)).
Fluorophore technology
One of the major challenges in modern fluorescence microscopy is the engineering of appro-priate dyes and the specific attachment to the biomolecule of choice. The properties required of chromophores for single-molecule imaging are demanding: the photostability in terms of lifetime and (absence of) blinking must be high, the conjugated molecular structure must be soluble and stable, and the fluorophore’s dimensions and physicochemical properties should not interfere with protein conformations and function. For sub-nanometre tracking high quantum yields and large Stokes shifts are especially important.
In general, three categories of probes can be differentiated: Fluorescent proteins, organic dyes and quantum dots. Being genetically encoded as fusion construct to the protein of interest, fluorescent proteins are labels with absolute specificity and represent a standard approach for
in vivo imaging. Limitations are their photostability and brightness, as well as the bulkiness
of the 25-kDa structure that potentially interferes with enzyme functionality. Organic dyes are significantly smaller and often display better photophysical properties. The commercial availability of dyes is enormous; brightness, stability, and solubility can be chosen with great flexibility. The major bottlenecks are the specificity and efficiency of the labelling chemistry and, for in vivo studies, the need for electroporation or alternative methods to introduce the dyes into the cell. Finally, quantum dots, fluorescent nanocrystals of 5-20 nm diameter, can be engineered in highly sophisticated ways, with extinction coefficients several times higher than those of organic dyes. Extremely high brightness and the resultant high signal-to-noise ratios allow nanometre-tracking of individual molecules (45). The large size, however, can influence the mobility and conformational flexibility of the labelled protein.
Fluorescent proteins
Fluorescent proteins (FPs) consist of a rigid b-barrel composed of 11 b-sheets that surround a central a-helix containing the chromophore (46). The naturally occurring variants have been extensively tuned in terms of brightness, emission range, photostability, monomeric charac-ter, and maturation rate (47, 48). Due to the strong autofluorescence of endogeneous cellular fluorophores (flavins, NADH, amino acids) at wavelengths below 500 nm, the development of red-shifted FPs is one central consideration for in vivo imaging.
Newly developed classes of FPs with photoconvertible or photoswitchable chromophores al-low super-resolution imaging even in a high-concentration environment, as only the
lim-ited fraction within the excitation field is switched on. Photoconvertible FPs such as Kaede, KikGR, Dendra and Eos are subject to a peptide-backbone cleavage step when illuminated with a 405 nm laser, leading to an enlargement of the conjugated system by an additional imidazole ring, which corresponds to a green-to-red shift in fluorescence (48, 49). The pho-toswitchable FP Dronpa has an excitation maximum at 503 nm, and can be switched off and on several times by strong 488 nm and weak 405 nm illumination, respectively. Alternatively, the green fluorescent Padron is switched on by blue excitation and off by UV light. The com-bination of those opposite switching behaviours allows two-color tracking in live cell imag-ing (50). In terms of photochemistry, crystal structures of Dronpa suggest that the cis-trans isomerization and protonation of the chromophore is responsible for the different fluorescent states (51).
Further progress in the design of fluorescent protein tags, especially far-red fluorescent as well as switchable probes in combination with novel microscopy techniques, will continue to provide powerful tools for in vivo imaging.
Organic dyes and their coupling to proteins
The main challenge in the use of organic dyes is a highly efficient and specific labelling reac-tion to the target protein. Several strategies exist for selective chemical tagging that can be basically subdivided into the introduction of a protein domain, a short peptide or a unique amino acid (52).
A successful method to specifically couple an organic dye to a protein is the fusion to a target protein of an additional protein domain that itself binds the organic dye tightly and selec-tively. Prominent protein-domain fusion constructs are the commercially available dehaloge-nase and alkylguanosine transferase tags (HaloTag and SNAP tag, respectively). The HaloTag technology takes advantage of a self-labelling step of a 33 kDa-sized dehalogenase enzyme. The reaction catalysed by this enzyme consists of 1) a nucleophilic displacement of a halide ion from an alkane chain that is transferred to an aspartate residue, 2) histidine catalysed hydrolysis, finally regenerating the aspartate. Mutagenesis of the active-site histidine residue locks the dehalogenase in step 1, allowing specific labelling with a customized fluorescent alkane moiety (53). The 20 kDa sized O6-alkylguanine-DNA alkyltransferase (hAGT) en-zyme transfers an alkyl group from guanosine derivatives to its active site cysteine residue, allowing for the subsequent covalent coupling of alkyl-modified fluorophores (54).
Smaller peptide tags are particularly advantageous when internal labelling positions are re-quired. The Tsien lab developed a biarsenic tagging technology that depends on the high affinity of thiols to arsenic (55, 56). The probes 4’, 5’-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) or the chemically similar resorufin-based ReAsH are non-fluorescent when bound to ethane dithiol (EDT), but fluoresce in green and red, respectively, when a tetracysteine se-quence CCXXCC replaces EDT. Another strategy relies on the incorporation of an aldehyde tagging sequence LCTPSR into the target protein (57). A co-expressed formyl-glycine-gen-erating enzyme converts the cysteine’s thiol group into an aldehyde that specifically reacts with hydrazide-functionalized molecules to a hydrazone. Other self-labelling tags are the hexa-histidine peptide or the Texas-red-binding aptamer, chelating with Ni-NTA-derivatized fluorophores or binding the Texas-red fluorophore with nano- to picomolar binding affinity
(58, 59).
Cysteines are usually less abundant in proteins and due to their high reactivity towards maleimide thioesters a popular target for in vitro labelling. If cysteine mutagenesis is not favourable because of limitations related to protein functionality, the introduction of unnat-ural amino acids, as pioneered by the Schultz lab, represents an alternative approach. Co-ex-pressed orthogonal tRNAs and aminoacyl tRNA synthetases incorporate a range of unnatural amino acids in response to amber stop codons or quadruplet codons (60-63).
Despite the intrinsic bottleneck of selectivity in labelling, the advantage of organic dyes lies in the nearly unlimited options for fluorescence characteristics. Not only are dyes available that cover the entire spectral range, but also many fluorescent compounds have been developed with properties that can be externally modified by optical inputs. For example, caged chro-mophores can be activated by UV light, and several cyanine dyes can be coupled to construct activator-reporter FRET pairs (64, 65).
2.2.2 Trapping and pulling at individual DNA molecules
Force spectroscopy methods are frequently applied for characterizing mechanical properties of biomolecules at the single-molecule level, such as topological changes in DNA molecules or force exertion by individual motor proteins. In the context of studying DNA replication at the single-molecule level, such techniques are often used to stretch the DNA substrate and to probe the mechanical consequences of replication on the DNA (conversion between single- and double-stranded DNA (8, 66), change in supercoiling (67)), or to observe the motion of proteins along DNA (68, 69). Detailed reviews about the instrumental designs can be found elsewhere (70-73).
In trapping techniques, one end of the biomolecule of choice is stably attached to a surface and the other one trapped with a magnetically or optically controlled bead or an AFM tip. Optical tweezers trap dielectric beads within a focused laser beam. The electromagnetic field polarizes the particle that is forced into the steep gradient at the focal spot. Spatial resolutions of down to 0.1 nm with sub-millisecond time resolutions are feasible by applying forces of about 0.1 to 100 pN (70). Magnetic traps have a slightly lower spatial resolution of about 2 to 10 nm, can apply forces over a large range from pico- to nano-Newton, and therefore are particularly useful in the measurement and manipulation of DNA topology. By attaching DNA on one end to a surface and on the other to a paramagnetic bead, the polymer is con-strained and can be accurately controlled and placed in a particular topological conformation with defined twist and writhe (74). Prominent topoisomerase experiments are performed on magnetically manipulated plectonemic DNA, as the ATP-dependent double-strand breaks remove two turns, thus changing the linking number by two (75). Flow-stretching techniques rely on the hydrodynamic dragging of one-end anchored polymers in a microfluidic device. DNA-bead tethers are for example useful in tracking length changes of the molecule during the time course of replication (76) (Figure 4).
Combining the strengths of fluorescence imaging and mechanical approaches, recent devel-opments have allowed the observation of DNA-based single-molecule fluorescence while
exerting well-defined stretching forces on the DNA template (77, 78). For example, Holliday-junction recombination events and conformational changes could be followed by creating FRET pairs within the four-stranded complex, tethered to an optical trap (79). The angstrom resolution of FRET signals combined with sub-pN forces in the optical trap established a highly controlled system for controlling and following conformations of DNA structures. Such hybrid techniques, allowing both the tracking of fluorescent molecules and the detec-tion of the chemomechanical reacdetec-tions, hold tremendous power in understanding the many facets of multi-protein machineries acting on DNA.
2.3 Replication machineries
Genomic DNA replication consists of three distinct phases: initiation, elongation and ter-mination. The complexity of cell-cycle timing, its coupling to DNA synthesis, and in general the molecular details of DNA synthesis vary tremendously amongst the taxonomic domains. However, the main principles of the replication machinery are conserved: Ring-structured replicative helicases encircle single-stranded DNA and couple the energy released from nu-cleotide hydrolysis to directional movement. The subsequent unwinding of the DNA provides a template for polymerases to synthesize the daughter strands by catalysing the coupling of an incoming nucleotide to the ribose 3’ hydroxyl group of the previously incorporated nu-cleotide. All known polymerases display this requirement of directionality: only DNA syn-thesis from the 5’ to the 3’ end allows for a continuation of synsyn-thesis (accompanied by the backwards removal of incorrectly incorporated nucleotides). With the antiparallel nature of double-stranded DNA, such a directional requirement for DNA synthesis results in a picture in which DNA is synthesized continuously on the so-called leading strand, with the lead-ing-strand DNA polymerase acting in the same direction as the helicase is moving, and with
S N
F
F
F
Flow-stretching Magnetic tweezers Optical tweezers
Figure 4: Force manipulation setups. DNA molecules are attached on one side to the coverglass surface and coupled
the lagging-strand DNA polymerase polymerizing in a discontinuous fashion, giving rise to short stretches of DNA named Okazaki fragments (Figure 5, A and B).
A special class of polymerases, primases, synthesize short oligo-ribonucleotide primers that are used as starting template for the lagging-strand DNA polymerase. The timing of the enzy-matic steps at the lagging strand, i.e. priming, utilization of the primer by the polymerase and its extension into an Okazaki fragment, are of importance for the orchestration of a coupled replication reaction: a process in which continuous synthesis on the leading strand is tightly coordinated with the discontinuous synthesis on the lagging strand.
Research on the replisome of the bacteriophage T4 initiated the idea of the trombone model that reconciles a symmetric replication fork, containing two DNA polymerases moving in the same direction, with the underlying asymmetry of the DNA template (80). The formation of a looped structure in the lagging strand reorients the polymerase while synthesizing an Okazaki fragment, until a release event triggers the recycling of the polymerase to the next Okazaki fragment. The DNA looping and the close proximity of the lagging-strand DNA polymerase to the replisome results in a short distance for the polymerase to be overcome after its release to utilize the next primer. The presence of replication loops is supported
Protein class T7 T4 E. coli Eukaryotes
Polymerase gp5 gp43
Clamp/ processivity factor thioredoxin gp453 2 PCNA
Clamp loader - gp444/62 / ‘x RFC
Helicase gp46 gp416 DnaB6 MCM2-7/CMG
Helicase loader - gp59 DnaC ORC licensing complex
ssDNA binding protein gp2.52 gp32 SSB4 RPA
Primase gp46 gp61 DnaG Primase
A B
C
α, δ, ε
Figure 5: Replisome proteins and fork architecture in viruses, bacteria and eukaryotes. A) The replication fork of
bacteriophage T7. Two polymerases gp5, each associated with an E. coli thioredoxin molecule, bind to the hexameric helicase/primase gp4. The primase domain of gp4 synthesizes short ribonucleotide primers that are handed over to the lagging-strand polymerase for elongation into Okazaki fragments. The unwound single-stranded regions of the template DNA are covered by gp2.5 proteins (adapted from Ref. (66)). B) The replication fork of E. coli. Two copies of the DNA polymerase holoenzyme are associated with b clamps on the leading and lagging strand. Three DnaG molecules associate with the DnaB helicase to synthesize primers on the lagging strand that is partly covered by SSB tetramers (adapted from Ref. (192)). C) Comparison of the replisome components in phage, E. coli and eukaryotes.
by several lines of evidence obtained from bacteriophage replication machineries generating Okazaki fragments of about 1000 to 2000 bp. In eukaryotes however, the much shorter Oka-zaki fragments (100 to 200 base pairs) make such a looping scenario less likely and certainly more difficult to observe.
In addition to the mechanistic demands placed on replication due to the antiparallel nature of duplex DNA, copying genomic stretches of DNA inside the cell comes with several other molecular challenges. Roadblocks such as nucleosomes need to be dealt with, the topology of the DNA needs to be controlled, and replication needs to be regulated and coordinated with other cellular activities such as DNA repair and recombination. As will be laid out in the remainder of this review, single-molecule biophysical techniques have begun to significantly contribute to our understanding of the molecular aspects of each of these processes. We will illustrate these efforts by starting with simple replication model systems, focusing on only the activities at the fork, followed by zooming out and considering the interplay of replication with topology, nucleosomes and the overall cell cycle.
2.3.1 Model systems for single-molecule studies
The main operating principles of the replisome are highly conserved across phages, bacteria and eukaryotes (Figure 5), although the involved enzyme classes are structurally not neces-sarily homologous. Replication complexes that are well understood in terms of their com-position, assembly and functioning are the ones of the bacteriophages T7 and T4, as well as that of E. coli. The much higher complexity of the eukaryotic replisome and of the cell-cycle checkpoints that regulate replication start and progression still requires further biochemical research in order to completely model the process of DNA duplication (81, 82). In the fol-lowing sections, we will discuss briefly the biochemical properties of these systems, before focussing on single-molecule studies.
Bacteriophage T7
As one of the simplest replication machineries in terms of the number of proteins involved, the bacteriophage T7 replisome has proven to be a powerful platform to study the coordina-tion of leading and lagging-strand synthesis, both at the ensemble and single-molecule level. Only four proteins (Figure 5 A) are needed to assemble a replication fork that proceeds with high processivity and stability, while also exhibiting remarkable dynamics in its interactions and composition. The DNA helicase/primase gene product 4, gp4, is responsible for both DNA unwinding and RNA primer deposition on the lagging strand. The N-terminal half of this bifunctional protein supports the primase activity. Faced away from the ds-ssDNA junc-tion, the N-terminal zinc-binding domain (ZBD) scans the single-stranded lagging strand as it is extruded by the C-terminal helicase domain. After recognition of a signal sequence, a tetraribonucleotide primer is synthesized by the RNA-polymerase domain (83). The ZBD remains associated with the primer and hands it off to the lagging-strand polymerase (84). The C-terminal helicase domain of gp4 hydrolyses dTTP to translocate along ssDNA in 5’ to 3’ directionality and displaces the complementary strand to unwind dsDNA. Gp4 exists as a hexamer as well as a heptamer in solution, but functions on ssDNA in its hexameric conformation (85, 86). As the T7 replisome lacks a helicase-loading protein in comparison to
other systems (see below), it is hypothesized that the loss of one subunit facilitates the loading mechanism (86). Alternatively or concomitantly, a loading site within the primase domain that interacts with the DNA may participate in the ring-opening mechanism required for loading on DNA (86, 87). The T7 DNA polymerase, a complex of gp5 with the E. coli thiore-doxin protein as processivity factor, synthesizes new DNA with one copy of the complex on the leading strand and one on the lagging strand. Gp5 on its own displays a processivity of only about 80 nt, but when bound to thioredoxin with a very low Kd of 5 nM (88), its binding lifetime to the primer-template, and thus its processivity, is increased ten-fold (89, 90). The activities of gp4 and gp5, unwinding and synthesis, are highly synergistic, so that a fully re-constituted T7 replisome achieves processivities of >17 kbp in leading-strand synthesis, while Okazaki fragments are generated in the lagging-strand loops approximately every 1-2 kbp (91). Finally, the ssDNA-binding protein gp2.5 binds and protects the transiently exposed single-stranded DNA on the lagging strand (91, 92). Beyond this classical ssDNA-binding role, gp2.5 is also important in mediating protein-protein interactions and regulating hand-off events at the replication fork (93, 94).
Bacteriophage T4
After its initial reconstitution in vitro by Alberts and coworkers in 1975 (95), the bacterio-phage T4 replisome has been one of the most intensively studied replication systems. Detailed knowledge exists of the various protein structures, protein-interaction sites and enzyme ki-netics, together forming an ideal basis for biophysical studies. A key property of the T4 system is its conceptual similarity to the replication systems of higher-order replisomes: like these, it contains ring-shaped clamp proteins that anchor the polymerases at the fork, clamp-loader proteins and helicase-loader proteins. The lower complexity, however, in terms of the total number of involved proteins or the regulation of replication initiation, has allowed the manip-ulation and study of its molecular mechanisms by single-molecule approaches.
The T4 replisome is composed of eight proteins (Figure 5 C), subdivided into the primosome (gp41 helicase, gp59 helicase loader, gp32 ssDNA-binding protein, gp41 primase) and the replicase/holoenzyme (gp43 polymerase, gp45 clamp, gp44/62 clamp loader) (96, 97). The hexameric helicase loader has a high affinity for gp32-coated DNA segments at replication forks and coordinates the loading of the hexameric helicase (98). Equimolar amounts of he-licase loader and hehe-licase were shown to be favourable for the hehe-licase unwinding activity, pointing to a 1:1 binding stoichiometry (99), analogous to the DnaB/DnaC complex in E. coli, as described below. The primase gp61 associates with the helicase on the lagging strand and synthesizes pentaribonucleotide primers to initiate Okazaki-fragment synthesis (100, 101). Reminiscent of the fused helicase/primase T7 gp4, gp61 employs maximal priming activity when present in 6:1 molar ratio to the hexameric helicase (102). As for T7, most likely a primer hand-off mechanism to either the ssDNA-binding protein or the polymerase exists that prevents the primer from melting (103). On both DNA strands, the polymerase gp43 associates with a trimeric sliding processivity clamp gp45 that prevents it from falling off the template and that is loaded by the gp44/62 clamp-loader complex (104). This pentameric complex is required to break up the ring-shaped clamp in order to thread the double stranded DNA through the clamp opening at the primer-template hybrid segment. The clamp-loader complex belongs to the class of AAA+ (ATPases Associated with diverse cellular Activities)
