Replicating Damaged DNA in Eukaryotes

DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.Before eukaryotic cells divide, the successful completion of DNA replication during S phase is essential to preserve genomic integrity from one generation to the next. During this process, the replication apparatus traverses in the form of bidirectionally moving forks to synthesize new daughter strands. Cells use several means to ensure faithful copying of the parental strands—first, by means of regulatory mechanisms a correctly coordinated replication apparatus is established, and second, a high degree of fidelity during DNA synthesis is maintained by replicative polymerases (Kunkel and Bebenek 2000; Reha-Krantz 2010). However, under several stressful circumstances, endogenously or exogenously induced, the replication apparatus can stall (Tourriere and Pasero 2007). Mostly, structural deformations in the form of lesions or special template-specific features arrest the replication process, activate checkpoint pathways and set in motion repair or tolerance mechanisms to counter the stalling (Branzei and Foiani 2009; Zegerman and Diffley 2009). Basic replication mechanism, its regulatory pathways and means to tolerate DNA damage are largely conserved across eukaryotic species (Branzei and Foiani 2010; Yao and O’Donnell 2010). Understanding the mechanisms involved may enable therapeutic intervention to several human conditions arising from an incomplete replication or from the inability to tolerate perturbations (Ciccia et al. 2009; Preston et al. 2010; Abbas et al. 2013). Enhanced replication stress has also been commonly identified in precancerous lesions, and the inactivation of checkpoint responses coping with this presumably oncogene-induced condition is considered necessary to establish the fully malignant phenotype (Bartkova et al. 2005; Negrini et al. 2010).It is not possible to treat this topic in a comprehensive manner in the allotted space; the reader is referred to excellent recent reviews for more details (Branzei and Foiani 2010; Jones and Petermann 2012). We will attempt to provide an overview of the various strategies that a eukaryotic cell invokes to avoid problems caused by replication stress related to DNA damage and, if problems arise, to tolerate damage without endangering the entire process of genome duplication. In this context, we will only give a brief outline of checkpoint responses that are discussed in more detail in Sirbu and Cortez (2013) and Marechal and Zou (2013). Also, a detailed discussion of translesion synthesis can be reviewed in Sale (2013).     

Mediators of Homologous DNA Pairing

Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms.Homologous recombination, the exchange of base pairing between homologous DNA molecules, is a central process for life (Morrical 2014). Not only does it create genetic diversity that sustains a population, it is essential at the cellular level for proper replication and maintenance of genomes (Wyman and Kanaar 2006). Given this central role in DNA metabolism, it is not surprising that the core reaction of homologous recombination is highly conserved among all kingdoms of life. The fundamental unit of this reaction is a DNA-strand-exchange protein, a small ATP-binding protein of ∼40 kDa. The DNA-strand-exchange protomers assemble head-to-tail in a right-handed helical filament around single-stranded (ss) DNA. This molecular assembly, programmed by the sequence of the bases of the bound DNA strand, recognizes homology in a double-stranded (ds) partner DNA molecule. Through protein-mediated manipulation of DNA structure and disassembly of protein–protein and protein–DNA interactions, exchange of base-pairing partners is achieved (Wyman 2011; Jasin and Rothstein 2013).It is essential that the DNA-strand-exchange reaction at the core of homologous recombination is regulated such that it is actively applied in specific and changing conditions. Inappropriate DNA rearrangements also need to be avoided where they would be disastrous rather than beneficial. Thus, there need to be tipping points at which the reaction can either be driven forward or reversed, an important feature required to attain quality control (Kanaar et al. 2008). To achieve intricate levels of regulation, reaction choreographers have evolved, which are often referred to as positive or negative recombination mediators or effectors (Daley et al. 2014). Interestingly, although the fundamentals of the DNA-strand-exchange reaction are the same for bacteriophages, bacteria, archaea, and eukaryotes (Maher et al. 2011; White 2011), it appears that the set of proteins that influences homologous recombination has expanded significantly during evolution of more complex life forms (Fig. 1). In this review, we focus on positive recombination mediators of the highly conserved DNA-strand-exchange protein proteins UvsX (bacteriophages), RecA (bacteria), and RAD51 (eukaryotes).Open in a separate windowFigure 1.The figure shows the increasing evolutionary complexity within the group of homologous recombination accessory factors that contribute to the formation and stability of the DNA-strand-exchange protein nucleoprotein filament in the key model organisms. Main homologous recombination steps (resection [Symington 2014], coating with ssDNA-binding protein [S], loading of the DNA-strand-exchange protein [R], and strand invasion) are shown schematically. Phylogenetic relationships between homologous proteins are indicated with solid lines in cases of well-supported orthology or broken lines when the exact evolutionary relationship is uncertain; x indicates no close homolog in a fully sequenced genome. Because phage, bacterial, and archaeal homologous recombination accessory proteins do not show detectably sequence similarity and have likely evolved independently, they are displayed as separate domains. Proteins that most closely meet the “mediator” definition criteria are indicated in bold. ∼ indicates the ability to promote annealing of protein-coated ssDNA, shared by UvsY, RecO, and RAD52, which is a remarkable example of convergent evolution emphasizing the universal usefulness of this biochemical activity.At the core of homologous recombination is a DNA-strand-exchange protein–ATP–ssDNA nucleoprotein filament (Wyman 2011). The positive recombination mediator proteins facilitate formation and functional activation of this molecular machinery. Specifically, in the original definition, recombination mediator proteins are described to facilitate loading DNA-strand-exchange proteins onto ssDNA that is coated by ssDNA-binding proteins (Beernink and Morrical 1999). Mediators influence the competition for ssDNA binding in favor of the filament forming DNA-strand-exchange protein. Mediators also influence the preferential binding of DNA-strand-exchange proteins to ssDNA in favor of the much more abundant dsDNA in cells. Although in biochemical assays the order of addition of protein can be controlled by the experimenter, with variable results for efficiency of assay outcome, in vivo exposed ssDNA will be rapidly and effectively bound by abundant, high-affinity ssDNA-binding proteins (Dickey et al. 2013). These proteins need to be prevented from binding or replaced by the DNA-strand-exchange protein to initiate homologous recombination. However, in part because of the semantic ambiguity of the term and the large number of factors affecting homologous recombination that still lack a clearly established biochemical function, the term mediator is often applied more generally in the literature. We prefer the term “homologous recombination accessory proteins” for this broader class of proteins, and will focus our discussion here on those mediators that fit the original definition when possible. A model for mediator-assisted sequential handover of ssDNA from the ssDNA-binding protein to the DNA-strand-exchange protein gradually emerged from studies of the slimmed down recombination systems, especially phage T4. The properties attributed to mediators include: ssDNA binding, interaction with the ssDNA-binding protein, interaction with the DNA-strand-exchange protein, and “filament stabilization” by affecting ATPase activity of the nucleoprotein filament (Liu et al. 2011a). This last feature, an effect on nucleotide exchange or ATPase activity by the nucleoprotein filament, became the template used to search for candidate mediators in higher organisms. Typically, the potential mediator proteins were then tested for the specific interactions and functions needed to promote DNA-strand-exchange protein filament formation and activation.     

Structural Studies of DNA End Detection and Resection in Homologous Recombination

DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies.All domains of life maintain genomes and ensure genetic diversity through homologous recombination (HR) or homology directed repair. HR is initiated by single unprotected DNA ends, which arise at collapsed replication forks and unprotected telomeres, or by DNA double-strand breaks (DSBs), which are products of ionizing radiation, reactive oxygen species, genotoxic chemicals, or abortive topoisomerase reactions (Sutherland et al. 2000; Aguilera and Gomez-Gonzalez 2008; Cadet et al. 2012; Mehta and Haber 2014). In special cellular states, programmed DSBs are introduced by endonucleases to initiate the generation of genetic variability by processes such as meiotic recombination of homologous chromosomes (Lam and Keeney 2014; Zickler and Kleckner 2014), V(D)J and class switch recombination to generate antibody diversity and yeast-mating-type switching (Gapud and Sleckman 2011; Haber 2012; Xu et al. 2012b). Failure to repair DSBs can lead to cell death or gross chromosomal aberrations, which in humans are a hallmark of cancer (Myung et al. 2001a,b; Hanahan and Weinberg 2011).Beside HR, DSBs can also be repaired by nonhomologous end joining (NHEJ). Although HR requires a template such as a sister chromatid or a homologous chromosome and is limited to S and G₂ phases of the cell cycle, NHEJ is template-independent and can occur in all cell cycle states. Indeed, the choice of pathways is to a significant extent not stochastic but a function of the cell cycle (Ferretti et al. 2013), with NHEJ being the predominant pathway in mammals outside of S phase. NHEJ is basically a ligation reaction of two DNA ends that are only minimally processed. Derivatives of NHEJ such as microhomology-mediated end joining (MMEJ) or alternative NHEJ (alt-NHEJ) require more substantial processing and may lead to the loss of genetic information. For recent reviews of NHEJ, which is not covered in detail here, please refer to, for example, Thompson (2012) and Chiruvella et al. (2013).HR has multiple steps and requires extensive processing of DNA ends (Symington 2014). First, the free DNA ends are recognized by DSB sensors, followed by 5′-3′ resection of the DNA ends. In eukaryotes and archaea, this step may be divided into initial short-range resection, after which MMEJ/alt-NHEJ can still occur, followed by processive long-range resection that commits the pathway to HR. The 3′ single-stranded DNA (ssDNA) filament, bound by the DNA strand exchange protein RecA/Rad51, pairs with the homologous sequence on the template and thus forms a D-loop. The 3′ tail serves as a primer for a repair polymerase and is extended by using the homologous strand as template, a process that “restores” the disrupted genetic information. Various pathways involve the displacement of the free strand, the capture of the second strand to form Holliday junctions, or the cleavage of the D-loop (Mehta and Haber 2014).In this review, we focus on structural aspects of the early steps in homologous recombination. Of particular interest is the Mre11-Rad50-Nbs1 (MRN) complex, which recognizes DSBs, performs initial resection, and sets off a DNA damage response (DDR) signaling network. We further discuss the nucleases and helicases that are involved in long-range resection. Recent reviews of later steps in HR, which are not covered here, have been published elsewhere (Amunugama and Fishel 2012; Chiruvella et al. 2013; Jasin and Rothstein 2013).     

Postreplicative Mismatch Repair

The mismatch repair (MMR) system detects non-Watson–Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences. Unsurprisingly, MMR malfunction brings about genomic instability that leads to cancer in mammals. But MMR proteins have recently been implicated also in other processes of DNA metabolism, such as DNA damage signaling, antibody diversification, and repair of interstrand cross-links and oxidative DNA damage, in which their functions remain to be elucidated. This article reviews the progress in our understanding of the mechanism of replication error repair made during the past decade.The mismatch repair (MMR) system is one of the key guardians of genomic integrity. Its malfunction leads to a substantial increase in spontaneous mutagenesis, illegitimate recombination, and cancer in mammals. MMR improves the fidelity of DNA replication by several orders of magnitude by excising sections of the nascent strand containing mispaired nucleotides. It is likely that MMR has evolved to carry out this function in order to ensure that daughter cells inherit an exact replica of the parental genome. But MMR also controls the fidelity of recombination by removing mispairs from heteroduplexes arising between donor and recipient strands and possibly even rejecting synapses between sequences that are too diverged. Indeed, the existence of MMR was first invoked in the 1960s to explain the unanticipated segregation of genetic markers in fungi and bacteria (for a comprehensive overview of the field, see chapter 12 in Friedberg et al. 1995). During the intervening 50 years, our understanding of MMR has made enormous progress; the main protagonists, as well as many “extras” that participate in this complex process, have been identified, initially in a series of genetic and biochemical experiments and later by sequence homology searches that were made possible by the high degree of evolutionary conservation of MMR. Analysis of the primary sequences of these polypeptides then helped to uncover their enzymatic activities that were confirmed by biochemical and structural studies. In vitro MMR assays using cell extracts and recombinant DNA substrates carrying single mismatches at defined positions led to the discovery of criteria required for efficient, strand-directional MMR. Finally, the Escherichia coli and the minimal human MMR systems could be reconstituted from purified recombinant proteins (Dzantiev et al. 2004). Despite this wealth of knowledge, however, we still lack detailed understanding of the molecular transactions that lead to successful repair of replication errors, and our notion of the role(s) of MMR proteins during recombination is highly speculative.Since the discovery of a link between its malfunction and cancer (for recent reviews, see Wimmer and Etzler 2008; Hewish et al. 2010), MMR has attracted a great deal of attention, and recent progress in our understanding of this pathway has been the subject of several reviews (Stojic et al. 2004; Kunkel and Erie 2005; Iyer et al. 2006; Jiricny 2006; Hsieh and Yamane 2008; Li 2008; George and Alani 2012; Peña-Diaz and Jiricny 2012). This article therefore provides only a brief overview of the MMR process and focuses primarily on the most recent insights into this complex pathway of DNA metabolism.     

Dormant Origins,the Licensing Checkpoint,and the Response to Replicative Stresses

Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.Replicating the large amount of DNA in eukaryotic cells is a complex task, requiring the activation of hundreds or thousands of origins spread throughout the genome. To maintain genetic stability, it is essential that during S phase genomic DNA is precisely duplicated, with no sections of DNA left unreplicated and no section of DNA replicated more than once. To prevent re-replication, cells divide the process of DNA replication into two non-overlapping phases. Prior to S phase, origins are licensed by the binding of minichromosome maintenance 2-7 (MCM2-7) double hexamers (Gillespie et al. 2001; Blow and Dutta 2005; Arias and Walter 2007). During S phase, these are activated as the core of the CMG (Cdc45-MCM-GINS) replicative helicase (Moyer et al. 2006; Ilves et al. 2010). Prior to the onset of S phase, licensing proteins are down-regulated or inhibited, so that no more origins can be licensed (Wohlschlegel et al. 2000; Tada et al. 2001; Li et al. 2003; Li and Blow 2005). One consequence of using this mechanism for preventing re-replication of DNA is that it is imperative that enough origins are licensed prior to S-phase entry, so that no regions of the genome remain unreplicated, even if some replication forks stall or some origins fail to initiate (Blow et al. 2011). Metazoan cells employ a licensing checkpoint to monitor that sufficient origins are licensed, inhibiting S-phase entry until this is established (Shreeram et al. 2002; Blow and Gillespie 2008).Here we review recent research showing how cells ensure complete genome duplication by licensing more replication origins in G1 than are normally used during S phase. The otherwise dormant replication origins become important for ensuring the completion of DNA replication if replication forks stall or are inhibited during S phase. We also review research showing how the licensing checkpoint ensures that a large enough number of origins are licensed before cells embark on S phase.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).     

Nuclear DNA Content Varies with Cell Size across Human Cell Types

Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity.Our understanding of the complexity of multicellular organisms, and the diverse cells of which they are comprised, has dramatically increased over the past several decades. Yet, we still lack an understanding of some of the most basic features of the cells that constitute multicellular organisms. For example, the number of different cell types in an organism, or the rate at which different cells grow, divide, and die, remain poorly understood (see Niklas 2015). But perhaps most important, we lack an understanding of the size and abundance of cells that constitute an organism (see Amodeo and Skotheim 2015). Cell size, in particular, affects virtually all structural and functional attributes of multicellular organisms, from the molecular level to the whole organism level.One key feature of organisms that may vary with cell size is the amount of nuclear DNA. Across species, genome size has long been known to correlate positively with cell and nuclear volume (Price et al. 1973; Szarski 1976; Olmo 1983). But within species, too, the nuclear DNA content of somatic cells has been shown in a few instances to increase with cell size in species such as Daphnia (Beaton and Hebert 1989) and Arabidopsis (Jovtchev et al. 2006). Such increases in nuclear DNA content can have important consequences for cell function, in general, and gene expression, in particular (Hancock et al. 2008; Lee et al. 2009; De Veylder et al. 2011; Marguerat and Bähler 2012).In the case of humans, substantial differences in DNA content have been observed in many human cell types. Indeed, since Watson and Crick described the structure of DNA, studies of healthy human tissues have reported the presence of polyploid cells (Winkelmann et al. 1987; Biesterfeld et al. 1994). The cell types in which this has been observed appear to have little in common, except that they are generally stable, fully differentiated cells (Winkelmann et al. 1987). Still, these observations have done little to change the traditional view that all healthy somatic cells in the human body hold the same characteristic quantity of DNA (∼7 billion base pairs) based on the long-standing principle of DNA constancy (Mirsky and Ris 1949). Deviations from the diploid quantity of DNA in humans, like other animals, are still often viewed as exceptional, tissue-specific, or indicative of pathology. A more synthetic view of differences in nuclear DNA content across human cell types may provide some clarity on these and other issues.In this review, we compile and analyze published data to examine the extent to which nuclear DNA content varies across diverse human cell types, and whether such variation is correlated with cell size. We then compare these results with previously reported relationships between nuclear DNA content and cell size within four other species. Finally, we compare these results with the relationships between diploid genome size and cell size observed across species in several broad taxonomic groups. These analyses suggest that systematic variation in nuclear DNA content is a more ubiquitous phenomenon in human cells than was previously appreciated. However, as we later discuss, the mechanisms underlying these patterns remain in question.     

Regulating DNA Replication in Plants

Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed.Faithful genome duplication during the S phase of the cell cycle uses strategies largely conserved in all eukaryotes (DePamphilis and Bell 2011) and is pivotal to preserve genome integrity. Genome duplication in dividing plant cells has the same requirements and constraints than in animal cells, including the strict rule of occurring once and only once every cell cycle. The initial discoveries of several basic biological processes were performed in studies with plant cells, e.g., transposons, telomeres, RNA interference, to cite a few. DNA replication in eukaryotes is not an exception. Pioneering work in the mid-1950s showed the semiconservative nature of chromosomal DNA replication in the common bean Vicia faba (Taylor et al. 1957). Since then, plant DNA replication studies have focused primarily on defining temporal patterns of DNA replication at the chromosomal level along the S phase. DNA fiber autoradiography was first used in plants to determine replicon size at the single molecule level (Nitta and Nagata 1976; Van’t Hof 1976) and genome organization in early and late replicon families (Van’t Hof et al. 1978; Van’t Hof and Bjerknes 1981; reviewed in Bryant 2010; Costas et al. 2011a).In this work, we will focus on: (1) plant DNA replication proteins (see also Supplemental Table 3 online); (2) plant DNA replication origins and their epigenetic landscape; (3) novel licensing mechanisms; (4) the relevance of DNA replication proteins in the control of the endoreplication cycle during plant development, and, finally; (5) a brief overview of duplication of plant DNA viruses.     

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2–7 in the pre-RC to an active DNA helicase, the Cdc45–Mcm–GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.The main events at the initiation step of DNA replication are the unwinding of double-stranded DNA and subsequent recruitment of DNA polymerases, to start DNA synthesis. Eukaryotic cells require an active DNA helicase to unwind the origin DNA. The core components of the replicative helicase, Mcm2–7, are loaded as a head-to-head double hexamer connected via their amino-terminal rings (Evrin et al. 2009; Remus et al. 2009; Gambus et al. 2011) onto Orc-associated origins, to form the pre-RC in late M and G₁ phases (see Bell and Kaguni 2013). However, Mcm2–7 alone does not show DNA helicase activity at replication origins. After the formation of the pre-RC, other replication factors assemble on origins, and the pre-initiation complex (pre-IC) is formed. The pre-IC is defined as a complex formed just before the initiation of DNA replication (Zou and Stillman 1998); in yeast, it contains at least seven additional factors: Cdc45, GINS, Dpb11, Sld2, Sld3, Cdc45, and DNA polymerase ε (Pol ε) (Muramatsu et al. 2010). The formation of the pre-IC is a prerequisite for the activation of the Mcm2–7 helicase; two additional factors, Cdc45 and GINS, associate with Mcm2–7 and form a tight complex, the Cdc45–Mcm–GINS (CMG) complex (Gambus et al. 2006; Moyer et al. 2006). This reaction requires components of the pre-IC and two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) (for reviews, see Labib 2010; Masai et al. 2010; Tanaka and Araki 2010). In this article, we summarize and discuss the manner via which the pre-IC is formed in yeasts and metazoa. Although there are some discrepancies, the process of formation of the pre-IC is conserved fairly well in these organisms.