Concurrent Replication and Methylation at Mammalian Origins of Replication

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and β-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.     

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).     

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.     

Initiation of Chromosomal DNA Replication in Mammalian Cell-Free Systems

Chromosomal DNA replication is a fundamental part of the cell division cycle of eukaryotes, and its disruption often leads to genome instability and cancer. A focus for regulation is the initiation of the first replication forks, marking the transition from G1 to S phase. Direct biochemical investigation of the establishment and further progression of chromosomal DNA replication in human somatic cell nuclei has become possible through a cell-free system that obeys cell cycle control. Since its development less than a decade ago, several modifications and adaptations of the original system have been reported, which have led to temporal resolution of replication complex assembly and to the identification of novel DNA replication factors. Here, I will review the different systems, highlight fundamental differences and unifying concepts, and discuss their potential for understanding chromosomal DNA replication in somatic mammalian cells.     

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH₂-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH₂ or the COOH termini of heterologous proteins.

We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

     

Molecular and Structural Transactions at Human DNA Replication Origins

The DNA replication origins of metazoan genomes are the sites of complex sequence-specific protein-DNA interactions determining their precise cycle of activation and deactivation, once only along each cell cycle. Some of the involved proteins have been identified (and particularly the essential six-protein Origin Recognition complex, ORC) thanks to their homology with the proteins identified in yeast. Whereas in the latter organism ORC has a specific affinity for an origin consensus, metazoan (and human) ORC shows no sequence specificity and no origin consensus is identifiable in their genomes. The modulation of topology around the origin sequence plays an essential role in the function of the human lamin B2 origin and the two topoisomerases interact specifically with it in a cell-cycle modulated way. The two enzymes are never present on the origin at the same time and compete, in different moments of the cell cycle, with the ORC2 subunit for the same sites in the origin area. The topoisomerases could give essential contributions to origin definition, as demonstrated by their capacity to bind specifically, in vitro the lamin B2 origin, either alone (topoisomerase I) or in a multi-protein complex (topoisomerase II). They also play critical roles in the origin activation-deactivation cycle, topoisomerase II probably contributing to attain and/or maintain a topological status fit for pre-replicative complex assembly and topoisomerase I allowing the topological adaptations necessary for initiation of bi-directional synthesis.     

Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells

Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.     

Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication

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Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.     

Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.     

Cell-Free Transmission and In Vivo Replication of Marek''s Disease Virus

Marek's disease virus recovered from the feather follicle of infected chickens was found to be infectious for chickens in cell-free preparations. The virus replicated in epithelial cells of the germinative layer of the feather follicle epidermis, producing both intranuclear and round or diffuse cytoplasmic inclusion bodies in the infected cells. It was found at this site 2 weeks postinoculation and prior to the development of tumor or other gross lesions. In the nucleus, many naked and a few enveloped herpesvirions were found, whereas the cytoplasm contained predominantly enveloped herpesvirions, which were usually within the cytoplasmic inclusion bodies. Approximately 80% of the extracellular virions were enveloped. Studies with both virulent and avirulent strains of the virus revealed a relationship between virulence, contagiousness, and replication of the virus in the feather follicle.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Replication Independent Formation of Extrachromosomal Circular DNA in Mammalian Cell-Free System

Extrachromosomal circular DNA (eccDNA) is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence- independent enzymes as human protein extract can produce mouse- specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg²⁺ and is enhanced by double strand DNA breaks.     

Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins

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Mapping Sites Where Replication Initiates in Mammalian Cells Using DNA Fibers

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller ( approximately 46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.