A Personal Reflection on the Replicon Theory: From R1 Plasmid to Replication Timing Regulation in Human Cells

Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases.     

Plasmid replication and partition in Escherichiacoli: is the cell membrane the key?

The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.     

Stochastic association of neighboring replicons creates replication factories in budding yeast

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.     

High resolution analysis of replication foci by conventional fluorescent microscopy. I. A study of complexity and DNA content of the foci

Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.     

Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.     

Stress responses and replication of plasmids in bacterial cells

Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.     

Strategies for helicase recruitment and loading in bacteria

DNA replication initiation in prokaryotes and eukaryotes requires the recruitment and loading of a helicase at the replication origin. To subsequently unwind the double-stranded DNA, the helicase must be properly positioned on the separated DNA strands. Several studies have revealed similarities and differences in the mechanisms used by different autonomously replicating DNA elements (replicons) for recruitment and activation of the appropriate helicase. Of particular interest are plasmid replicons that are adapted for replication in diverse bacterial hosts and are therefore intriguingly able to exploit the helicases of distantly related bacterial species. The different molecular mechanisms by which replicons recruit and load helicases are only just beginning to be understood.     

Diversity, biology and evolution of IncQ-family plasmids

Plasmids of IncQ-family are distinguished by having a unique strand-displacement mechanism of replication that is capable of functioning in a wide variety of bacterial hosts. In addition, these plasmids are highly mobilizable and therefore very promiscuous. Common features of the replicons have been used to identify IncQ-family plasmids in DNA sequence databases and in this way several unstudied plasmids have been compared to more well-studied IncQ plasmids. We propose that IncQ plasmids can be divided into four subgroups based on a number of mutually supportive criteria. The most important of these are the amino acid sequences of their three essential replication proteins and the observation that the replicon of each subgroup has become fused to four different lineages of mobilization genes. This review of IncQ-family plasmid diversity has highlighted several events in the evolution of these plasmids and raised several questions for further research.     

Visualising chromosomal replication sites and replicons in mammalian cells

The precise regulation of DNA replication is fundamental to the preservation of intact genomes during cell proliferation. Our understanding of this process has been based traditionally on a combination of techniques including biochemistry, molecular biology and cell biology. In this report we describe how the analysis of the S phase in mammalian cells using classical cell biology techniques has contributed to our understanding of the replication process. We describe traditional and state-of-the-art protocols for imaging sites of DNA synthesis in nuclei and the organisation of active replicons along DNA, as visualised on individual DNA fibres. We evaluate how the different approaches inform our understanding of the replication process, placing particular emphasis on ways in which the higher order chromatin structures and the spatial architecture of replication sites contribute to the orderly activation of defined regions of the genome at precise times of S phase.     

Equations for measuring the rate of DNA chain growth and replicon size by density labeling techniques

The effects of initiation, termination and clustering of replicons have been considered in the development of equations which are used to measure DNA chain elongation in mammalian cells by density gradient techniques. These equations can be used to determine the average replicon size as well as the rate of chain elongation. Experimental data from WI-38 and HeLa cells are presented which show the applicability of these methods. Several models of replication initiation and termination have been developed. Some of these models can be eliminated experimentally and the experimental results are consistent with the notion that neighboring replicons are initiated simultaneously or sequentially with short (～-0·5 min) interinitiation time.     

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.     

Effect of puromycin on DNA replication in Chinese hamster cells

We have used autoradiography to examine the effect of puromycin on DNA replication in Chinese hamster cells aligned by treatment with fluorodeoxy-uridine. In the absence of puromycin the patterns of replication are consistent with those obtained previously by others (Cairns, 1966; Huberman &; Riggs, 1968). In particular, replication occurs in tandem clusters of replicons but not all replicons in a cluster appear to be activated at the same time.Puromycin decreases the over-all rate of synthesis of DNA per cell, but does not inhibit chain elongation as visualized in autoradiograms. It is suggested that puromycin inhibits the initiation of replication of replicons not yet activated. Puromycin prevents the joining of short stretches of radioactive DNA into longer pieces. This may be due to the inability to activate a few late replicating units within a cluster of replicons.     

Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.     

Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance

We have identified and characterized a genetic function (designated par, for partition) that is required for stable maintenance of plasmids within exponentially growing cell populations. This function, which accomplishes the active distribution of plasmid DNA molecules to daughter cells, has been localized within the pSC101 plasmid to a 270 bp segment adjacent to the replication origin. The par locus, which appears to be functionally equivalent to the centromere of eucaryotic cells, is able to rescue unstable pSC101-derived replicons or an unrelated par- P15A-derived multicopy replicon in the cis, but not the trans, configuration. It is independent of copy number control and dose not specify plasmid incompatibility. Furthermore, it is not associated directly with plasmid replication functions.     

Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites

Mammalian cells are constantly threatened by multiple types of DNA lesions arising from various sources like irradiation, environmental agents, replication errors or by-products of the normal cellular metabolism. If not readily detected and repaired these lesions can lead to cell death or to the transformation of cells giving rise to life-threatening diseases like cancer. Multiple specialized repair pathways have evolved to preserve the genetic integrity of a cell. The increasing number of DNA damage sensors, checkpoint regulators, and repair factors identified in the numerous interconnected repair pathways raises the question of how DNA repair is coordinated. In the last decade, various methods have been developed that allow the induction of DNA lesions and subsequent real-time analysis of repair factor assembly at DNA repair sites in living cells. This combination of biophysical and molecular cell biology methods has yielded interesting new insights into the order and kinetics of protein recruitment and identified regulatory sequences and selective loading platforms for the efficient restoration of the genetic and epigenetic integrity of mammalian cells.     

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.