A journey through the microscopic ages of DNA replication

Scientific discoveries and technological advancements are inseparable but not always take place in a coherent chronological manner. In the next, we will provide a seemingly unconnected and serendipitous series of scientific facts that, in the whole, converged to unveil DNA and its duplication. We will not cover here the many and fundamental contributions from microbial genetics and in vitro biochemistry. Rather, in this journey, we will emphasize the interplay between microscopy development culminating on super resolution fluorescence microscopy (i.e., nanoscopy) and digital image analysis and its impact on our understanding of DNA duplication. We will interlace the journey with landmark concepts and experiments that have brought the cellular DNA replication field to its present state.


The (very) early years
Long after water-filled glass bowls were used to read small letters (Singer 1914), a simple single lens microscope started the microscopic revolution (Bacon 1267) (see Table 1 and Fig. 1). Spurred throughout the ages by accidental inventions (van der Aa 1851), leaps by Galileo (Galilei 1610), and Hooke (1665), it was not until Carl Zeiss started to mass-produce microscopes in 1847 that DNA observation started to take off. Simultaneously, Mendel studied 29,000 pea plants (1866) and Haeckel postulated the containment of hereditary traits in the nucleus (1866) (Dahm 2008;Haeckel 1866), while Miescher put the microscope to good use and purified the nuclei and observed DNA (Miescher 1871). Köhler's gamechanging illumination technique (Köhler 1893) helped to perfect Zeiss UV-microscope together with Siedentopf in 1908. In 1927, shortly after Levene described the nucleic acid structure (1919), Koltsov postulated the semiconservative replication idea (Soyfer 2001).
The race down to the DNA structure and duplication Phase contrast microscopy (Zernike 1955) and DNA X-ray diffraction images (Astbury 1947) Franklin, 1952, BPhoto 52^) lead to fantastic images, new discoveries, and the description of the double helix DNA structure (Watson and Crick 1953). Meselson and Stahl ingeniously demonstrated the semiconservative mode of DNA replication (Meselson and Stahl 1958). The theoretical description of a confocal microscope and the first practical application of a Nipkow disk in microscopy (Egger and Petráň 1967;Petráň et al. 1968) were clear landmarks of the microscopy revolution.
Radioactive labeling and autoradiography allowed Cairns to observe DNA unwinding and the replication fork (Cairns 1963), and Huberman and Riggs observed similar replication structures in mammalian chromosomes (Huberman and Riggs 1966) and Okazaki described the lagging strand synthesis and Bits^fragments Okazaki and Okazaki 1969;Sugimoto et al. 1969;Sugimoto et al. 1968). Water filed glass bowls to read small letters (Singer 1914) 1267 The first simple microscope (Bacon 1267) 1590 Accidental discovery of the compound microscope with two (or more) lenses by Zacharias Janssen BReplicate in a semiconservative fashion using each strand as a template^ (Soyfer 2001(Soyfer ) 1932 Discovery of the electron microscope (Knoll and Ruska 1932a;Knoll and Ruska 1932b) 1947 DNA X-ray diffraction images (Astbury 1947(Astbury ) 1953 X-ray diffraction BPhoto 51^ (Watson andCrick 1953) 1953 Discovery of the double-helix DNA structure (Watson andCrick 1953) 1953 Discovery of phase contrast microscopy (Zernike 1955(Zernike ) 1958 Confirmation of the semiconservative DNA replication model (Meselson andStahl 1958) 1957 Discovery of the confocal microscope (Minsky 1961(Minsky ) 1962 Extraction, purification, and properties of GFP (Shimomura et al. 1962) 1963 DNA unwinding for replication and Breplication fork^ (Cairns 1963(Cairns ) 1966 Autoradiography of chromosomal DNA fibers from Chinese hamster cells. (Huberman andRiggs 1966) 1966 On the mechanism of DNA replication in mammalian chromosomes (Huberman andRiggs 1968) 1967 First practical application of the BNipkow disk^in confocal microscopy (Egger and Petráň 1967;Petráň et al. 1968Petráň et al. ) 1968 Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains.  Mechanism of DNA chain growth, II. Accumulation of newly synthesized short chains in E. coli infected with ligase-defective T4 phages. (Sugimoto et al. ) 1969 Duration of the cell cycle (Van Dilla et al. 1969 Mechanism of DNA chain growth, III. Equal annealing of T4 nascent short DNA chains with the separated complementary strands of the phage DNA (Sugimoto et al. 1969(Sugimoto et al. ) 1969 Mechanism of DNA chain growth. IV. Direction of synthesis of T4 short DNA chains as revealed by exonucleolytic degradation.  1972 Bidirectional Replication of Simian Virus 40 DNA (Danna and Nathans 1972) 1974-1979 Fork speed, replication speed, and replicon sizes (Kriegstein and Hogness 1974;Taylor 1977;Taylor and Hozier 1976;Wilson and Wilson 1975;Yurov 1977;Yurov 1978;Yurov 1979;Liapunova 1977) 1975 Continuous cultures of fused cells secreting antibody of predefined specificity. 1986 Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus (Nakamura et al. 1986(Nakamura et al. ) 1989 Three distinctive replication patterns (Nakayasu andBerezney 1989) 1992 Dynamic organization of DNA replication in mammalian cell nuclei spatially and temporally defined replication of chromosome (O'Keefe et al. 1992) 1992 Progression of DNA synthesis (Rizzoli et al. 1992) 1993 Structured Illumination Microscopy (SIM) (Bailey et al. 1993(Bailey et al. ) 1994 Green fluorescent protein as a marker for gene expression (Chalfie et al. 1994(Chalfie et al. ) 1994 4pi microscope (Hell 2003;Hell et al. 1994)  1994 Alignment and sensitive detection of DNA by a moving interface (Bensimon et al. 1994) 1997 The replication origin decision point is a mitogen (Wu and Gilbert 1997) 1997 Dynamic molecular combing: stretching the whole human genome for high-resolution studies. (Michalet et al. 1997(Michalet et al. ) 1998 Replicon clusters are stable units of chromosome structure evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells (Jackson andPombo 1998) 1999 The spatial position and replication timing of chromosomal domains are both established in early G1 phase (Dimitrova and Gilbert 1999) 1999 Single molecule analysis of DNA replication. (Herrick and Bensimon 1999) 2000 Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci (Berezney et al. 2000) 2000 Dynamics of DNA replication factories in living cells (Leonhardt et al. 2000) 2000 DNA replication at high resolution (Keck and Berger 2000) 2000 Mechanisms of DNA replication (Davey and O'Donnell 2000) 2001

Eukaryotic origins 2001
Repression of origin assembly in metaphase depends on inhibition of RLF-BCdt1 by geminin (Tada et al. 2001) 2001 Visualization of DNA replication on individual Epstein-Barr Virus episomes (Norio and Schildkraut 2001) 2002 DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters (Sporbert et al. 2002) 2002 DNA replication and chromatin (Gerbi and Bielinsky 2002) 2002 Initiation of DNA replication in multicellular eukaryotes (Gerbi et al. 2002) 2003 Sequence-independent DNA binding and replication initiation by the human origin recognition complex (Vashee et al. 2003) 2003 The 'ORC cycle': a novel pathway for regulating eukaryotic DNA replication (DePamphilis 2003(DePamphilis ) 2004 Stable chromosomal units determine the spatial and temporal organization of DNA replication (Sadoni et al. 2004) 2004 DNA replication and DNA repair mechanisms most of the replication machinery is also used in DNA repair. (Sancar and Lindsey-Boltz 2004) 2005 Preventing rereplication (Blow and Dutta 2005) 2005 PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins (Sporbert et al. 2005) 2005 Eukaryotic origins of DNA replication: could you please be more specific? Walter 2005) 2006 Origin selection and silent origins (Patel et al. 2006) 2006   Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs. (Besnard et al. 2012) 2012 Targeted manipulation of heterochromatin rescues MeCP2 Rett mutants and re-establishes higher order chromatin organization. Replication dynamics: biases and robustness of DNA fiber analysis. (Técher et al. 2013) Always look on the bright side Along came Aequorea victoria green fluorescent protein (Shimomura et al. 1962) and brought light into darkness. Where audioradiography once ruled (Huberman and Riggs 1966;Huberman and Riggs 1968;Taylor et al. 1957), immunofluorescence labeling of fixed cells with monoclonal antibodies to modified nucleotides incorporated into newly synthesized DNA took the stage (e.g., Aten et al. 1992;Cardoso et al. 1993;Jackson and Pombo 1998;Jaunin et al. 1998;Ma et al. 1998;Mazzotti et al. 1990;Nakamura et al. 1986;Raska et al. 1989;Raska et al. 1991) only to be outshined by live cell microscopy of fluorescent fusion proteins (Cardoso et al. 1997;Leonhardt et al. 2000). Cell cycle duration (Van Dilla et al. 1969), fork speed, replication rate, and replicon sizes (Kriegstein and Hogness 1974;Taylor 1977;Taylor and Hozier 1976;Wilson and Wilson 1975;Yurov 1977;Yurov 1978;Yurov 1979;Yurov and Liapunova 1977) were all unearthed from the dark. In parallel, the first affordable home computers made digital image analysis possible through the help of Wayne S. Rasband who developed the milestone in image analysis ImageJ (then, NIH Image) in 1987 (Schneider et al. 2012).
Extensive microscopic analysis in fixed cells followed and provided a spatiotemporal description of replication sites (replication foci; see Fig. 2) in cells throughout S-phase (Nakamura et al. 1986) along with the three main distinctive early, mid, and late S-phase replication foci patterns (Jackson  Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. (Ticau et al. 2015) 2015 Single-molecule visualization of MCM2-7 DNA loading: seeing is believing. (Chistol and Walter 2015) 2015 High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins. (Comoglio et al. 2015) 2015 The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level.

Bacon
Simple microscope

Ruska
Electron microscope

Bensimon
Alignment and detection of DNA

Sporbert
De novo assembly at adjacent origins

Gao
DeOri: database of eukaryotic origins

Nakamura
Structural organization of replicons  Starting point of DNA replication, the replication origin (ori), and the region replicated from a single origin displayed in the bottom row. Each replicon is replicated by two replication machineries (magenta), composed of various replication proteins, magnified in the bottom left corner. Adapted from Chagin et al. 2010) and Pombo 1998; Mills et al. 1989;Nakayasu and Berezney 1989). Alongside, replication origins (Burhans et al. 1990;Burhans et al. 1991) were also reported.
The quest to replicate the genome DNA loops (see Fig. 2) and their Bfunctional^attachments to active transcription units were shown as chromatin organizers during mitosis (Jackson et al. 1992), and replication factories were proposed as clusters of DNA replication sites organized by the nucleoskeleton (Hozák et al. 1993). Molecular combing, refined DNA fiber analysis, and sensitive detection of DNA (Bensimon et al. 1994) opened the door to whole genome stretching and high-resolution studies (Michalet et al. 1997). It allowed analysis of single DNA molecules undergoing replication (see Fig. 2) in a much greater resolution (Herrick and Bensimon 1999) than ever before. Stable replicon clusters were also described as a basis for effective activation and propagation of genome replication during S-phase (Jackson and Pombo 1998) and regulation of replication timing (Dimitrova and Gilbert 1999). Studies on DNA replication proteins (see Fig. 2) using livecell fluorescence microscopy produced time lapse movies of replication factories and elucidated basic principles of their dynamic assembly-disassembly behavior (Leonhardt et al. 2000). Different regulatory levels were shown to be necessary to initiate and regulate DNA replication. Not only the chromatin structure, nuclear, and chromosomal locations but also origin recognition complex (ORC) and a whole bunch of other factors were found to define start sites of replication (DePamphilis 2003;Gerbi and Bielinsky 2002;Gerbi et al. 2002;Sasaki and Gilbert 2007).

Factories full of dominos
In addition to the Bfactory model^ (Hozák et al. 1993), more dynamic models ensued (Sadoni et al. 2004;Sporbert et al. 2002) whereby replication at one site induces domino-like activation of neighboring origins, without the need to postulate pre-determined clusters of replicons. The combination with an earlier model postulating that origins of replication would be licensed only during mitosis and this license to replicate would be revoked after one round of replication (Blow and Dutta 2005;Blow and Laskey 1988) elegantly demonstrated how DNA is completely duplicated once, and only once, during each cell cycle. Despite Cvetic wishing for Beukaryotic origins of DNA replication to please be more specific^ (Cvetic and Walter 2005), DNA replication origins in higher eukaryotes have been at best elusive. Nonetheless, as a whole, DNA replication is a very robust mechanism and stalled forks can be reactivated or reactivate neighboring origins to close all gaps and provide a perfect copy of billions of nucleotides at every cell division (Langston and O'Donnell 2006;Patel et al. 2006).

The ever elusive origin
The hunt for the elusive consensus motif of DNA replication origins continued with genome-wide high throughput mapping of potential origins and next-generation sequencing methods (Besnard et al. 2012;Cadoret et al. 2008;Cayrou et al. 2012;Dellino et al. 2013;Karnani et al. 2010;Lucas et al. 2007;Martin et al. 2011;Mesner et al. 2013;Mesner et al. 2011;Mukhopadhyay et al. 2014;Picard et al. 2014;Valenzuela et al. 2011) but stalled without a conclusive definition of the mammalian origin of replication. Correlations with specialized DNA structures (e.g., G-quadruplexes) and many others have been suggested but there seems not to be a simple solution and potentially there is no need to have one.
Studies into the epigenomic landscape, epigenetic control of DNA replication, and higher order chromatin organization (Casas-Delucchi and Cardoso 2011;Casas-Delucchi et al. 2012) have provided a link of epigenetic modifications (in particular, histone acetylation level) and temporal control of DNA replication origin firing.
Altogether, even Hyrien's BPeaks cloaked in the mist,^all out approach was not able to identify possible origins by similarities in thousands of microarrays and/or next-generation sequencing data, suggesting origins form at unspecific DNA sites, but are suppressed by ongoing transcription (Hyrien 2015), which is highly correlated with histone acetylation.
To go where no one has gone before: beyond the Abbe limit Meanwhile, the microscopy arms race to and beyond the diffraction limit calculated by Abbe continued with the Structured Illumination Microscopy (SIM) (Bailey et al. 1993), the 3D-SIM (Gustafsson et al. 2008) and the stimulated emission depletion (STED) (Schmidt et al. 2008).
The first attempts to label dating back to 1986 (Nakamura et al. 1986) and quantify replication sites in cells yielded numbers on the low hundreds (see Fig. 3). A decade later with the advent of digital imaging and computational image analysis tools, these numbers grew to around one thousand (Berezney et al. 1996;Fox et al. 1991;Jackson and Pombo 1998;Ma et al. 1998), where they remained for several years (see Fig. 3). Such numbers of replication sites were compatible with a concept of clusters of replicons activated together and, thus, visualized together.
The dramatic increase in spatial resolution made possible with the new super-resolution microscopy techniques (fluorescence nanoscopy) enabled the visualization, for the first time, of smaller replication structures (Baddeley et al. 2010;Cseresnyes et al. 2009;Ligasová et al. 2009). It was now possible to resolve structures well below the Abbe limit, down to 30 nm and smaller. Nanoscopy (Gustafsson et al. 2008;Hell 2003;Hell et al. 1994) is in full swing and let us go where no one has gone before: beyond the Abbe limit. This, on the other hand, created another level of demand upon image analysis tools.

BMyths^confirmed!
The stage was now set to try and unveil the units of genome replication, i.e., the replicons and their associated machinery, the replisome, in cells.
From the earlier studies using light nanoscopy techniques (Baddeley et al. 2010;Cseresnyes et al. 2009) as well as electron microscopy (Koberna et al. 2005), suitable computational image analysis protocols were developed (Chagin et al. 2015). These combined efforts led to a further increase in the numbers of replication sites measured in cells (see Fig. 3), which was now finally compatible and fitting with the predicted numbers of replicons needed to duplicate the genome in human cells Löb et al. 2016).
The microscopic information age had arrived. Previous efforts by Shaw et al. (2010), together with measurements throughout the years culminating on the visualization and quantification of individual replicons in cells in 4D, all supported by 3D-SIM imaging  were all combined in a minimalistic but comprehensive 4D replicon simulation model (Löb et al. 2016) displaying previously published replication polarity gradients, replication timing profiles, N/U domains, topologically associating domains, and timing transition regions (Audit et al. 2013;Baker et al. 2012;Chen et al. 2010;Hyrien et al. 2013;Pope et al. 2014).

Journey into the future
Future work should aim to bridge the ever-increasing genomewide population data, with single molecule and single-cell microscopic data. Novel ways to combine and relate these very different types of information should be developed to get the highest spatial together with the highest temporal resolution without compromising the data on variability between single cells.
Importantly, the available models should be put to work to predict and test genome replication in different cell types and species and under different stress conditions. This would unleash the value of the existing models and lead us into the in silico DNA replication era.

Conflict of interest
The authors declare that they have no conflict of interest. Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.