Initiation of DNA replication in mammalian cells and its inhibition by reovirus infection

We have studied the initiation of DNA replication in mammalian cells in tissue culture, using DNA fiber autoradiography to analyze initiation events occurring during 10 or 30-minute [³H]thymidine labeling periods. The mean distance between initiation sites varies in cells from different mammalian species. In mouse L cells, functioning initiation sites are distributed in clusters. The modal interval between individual sites is 40 to 50 μm, and the sites do not appear to occur at regular intervals. Initiation events appear partially synchronized in autoradiograms from DNA fibers spread over any one microscopic field of the autoradiographic slide (0.16 mm²), suggesting that such events occur in bursts over topographically contiguous regions of DNA. In cells infected with reovirus, a cytocidal RNA virus that markedly inhibits overall DNA replication in infected L cells, the distance between initiation sites labeled during a ten-minute pulse is increased. This indicates that the frequency of initiation events over localized regions of DNA is reduced by reovirus infection.     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing

Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.     

Eukaryotic origins of DNA replication: could you please be more specific?

Initiation of eukaryotic DNA replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in eukaryotic genomes.     

DNA replication origin interference increases the spacing between initiation events in human cells

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.     

Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-β. The ratio of ~0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to λ-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-β origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-β′). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-β, ori-β′, and ori-γ), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

Initiation of eukaryotic DNA replication: conservative or liberal?

The mechanism for initiation of eukaryotic DNA replication is highly conserved: the proteins required to initiate replication, the sequence of events leading to initiation, and the regulation of initiation are remarkably similar throughout the eukaryotic kingdom. Nevertheless, there is a liberal attitude when it comes to selecting initiation sites. Differences appear to exist in the composition of replication origins and in the way proteins recognize these origins. In fact, some multicellular eukaryotes (the metazoans) can change the number and locations of initiation sites during animal development, revealing that selection of initiation sites depends on epigenetic as well as genetic parameters. Here we have attempted to summarize our understanding of this process, to identify the similarities and differences between single cell and multicellular eukaryotes, and to examine the extent to which origin recognition proteins and replication origins have been conserved among eukaryotes. Published 2000 Wiley-Liss, Inc.     

Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene

Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

The origin recognition complex marks a replication origin in the human TOP1 gene promoter

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.     

AT-rich region and repeated sequences - the essential elements of replication origins of bacterial replicons

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.     

The structure of the initiation complex at the replication origin, oriC, of Escherichia coli.

Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA. The AT-rich region in the left part of oriC and the start site region in the right part of oriC. Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region. We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function. An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC. DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo. We propose a specific compact nucleoprotein structure for the initiation complex.     

Initiation of DNA replication in higher eukaryotes

In this review, of problems concerning initiation of DNA replication in higher eukaryotes is discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.     

Attachment of DNA loops to an artificial matrix does not affect the origin of replication initiation in early development of African clawed frog

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Initiation of DNA Replication in Cells of Higher Eukaryotes

In this review, the problems concerning initiation of DNA replication in higher eukaryotes are discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.