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Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus* 1
Abstract
In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with [3H]thymidine at the limited resolution level of autoradiography. By immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.
... These replication foci form distinct spatial patterns, characterized by the dynamic intra-nuclear distribution of the replication signals during S-phase progression (5)(6)(7)(12)(13)(14). In metazoan cells, three main patterns are observed, at early (I), mid (II) and late (III) S-phase, corresponding to the replication of euchromatin, facultative and constitutive heterochromatin, respectively (10,15,16). ...
... Hence, loop structures, potentially mediated by cohesins or functionally related proteins (25,26), represent the DNA element that defines replicons as functional unit in the DNA replication context (reviewed in (27)). Labeling cells with modified nucleotides revealed that the replicon clusters observed on DNA fibers become visible as the before-mentioned replication foci in interphase nuclei (15). With higher optical resolution levels, the number of replication foci measured in cells increased and each replication nanofocus in somatic mammalian cells was shown to be equivalent to a replicon unit (28,29). ...
... Replication patterns are a direct visual representation of the spatial organization and temporal order of DNA replication and, in somatic cells, have been shown to reflect the chromatin organization level (10,15,16). DNA replication timing profiles (RT-profiles) from large cell populations (43,65-68) revealed distinct replication domains (1.5-2.5 Mb), that exhibit sharp boundaries between neighboring domains with different replication timing, alternating along individual chromosomes (43). ...
To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.
... Spatio-temporal genome replication patterns correlate with the 3D genome organization and underlying chromatin structure [1,2]. Earlier studies of genome replication progression in mouse somatic cells using nucleotide analogs (5-bromo-2 ′ -deoxyuridine (BrdU) and 5-ethynyl-2 ′ -deoxyuridine (EdU)) coupled with sequencing analysis or fluorescence microscopy showed the correlation between DNA replication timing and increasing chromatin compaction [3,4]. ...
... The details of the cell lines used are described in Table 1. All cells were grown in a humidified atmosphere of 5% CO 2 [13] hiPSC A4 Homo sapiens iPSC from human neonatal foreskin fibroblast (HFF1) Male [14] hiPSC B4 Homo sapiens iPSC from human neonatal foreskin fibroblast (HFF1) Male [14] hTERT RPE1 Homo sapiens hTERT immortalized retinal pigment epithelial cell Female [15] BJ-5ta Homo sapiens hTERT immortalized foreskin fibroblasts Male [15] For live-cell time-lapse microscopy, cells were transfected with the plasmid pENeGF-PCNAL2mut (pc0653, https://www.addgene.org/167564/ accessed on 25 January 2024) [4]. ...
DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and differentiated cells. Using single-cell microscopic approaches, we map the spatio-temporal genome replication as a function of chromatin marks/compaction level. Furthermore, we mapped the replication timing of subchromosomal tandem repeat regions and interspersed repeat sequence elements. Albeit the majority of these genomic repeats did not change their replication timing from pluripotent to differentiated cells, we found developmental changes in the replication timing of rDNA repeats. Comparing single-cell super-resolution microscopic data with data from genome-wide sequencing approaches showed comparable numbers of replicons and large overlap in origins numbers and genomic location among developmental states with a generally higher origin variability in pluripotent cells. Using ratiometric analysis of incorporated nucleotides normalized per replisome in single cells, we uncovered differences in fork speed throughout the S phase in pluripotent cells but not in somatic cells. Altogether, our data define similarities and differences on the replication program and characteristics in human cells at different developmental states.
... Due to the sizes of mammalian genomes and the absence of defined origin sequences, no comprehensive assessment has been made of origin firing kinetics throughout S phase, but detailed studies have been made of the number of origins fired at the onset of S phase. This is achieved by counting replication foci labelled by BrdU incorporation into nascent DNA (Nakamura et al., 1986). The co-localisation of these BrdU foci with replication proteins such as PCNA and RPA supports their being authentic sites of DNA replication (Kill et al., 1991;Krude, 1995). ...
... At the start of S phase, each discernable focus is thought to represent an individual cluster of replicons. The intensity of such foci com pared to mitochondrial DNA indicates that most foci must contain more than one replicon (Nakamura et al., 1986); furthermore, early studies by fibre autoradiography revealed tandem arrays of up to 4 replicons of similar size, (Hand, 1975;Hand, 1977). More recently, this clustered arrangement of replicons has been supported by more detailed analyses of DNA fibres from a variety of mammalian cell lines. ...
Eukaryotic cells respond to DNA damage within S phase by activating an intra-S phase checkpoint; a response which includes reducing the rate of DNA synthesis. In yeast cells this occurs via a checkpoint-dependent inhibition of origin firing and stabilisation of ongoing forks, together with a checkpoint- independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. This work describes DNA fibre labelling strategies that offer a quantitative assessment of rates of replication fork movement, origin firing and fork stalling throughout the genome by examining large numbers of individually labelled replication forks. It shows that exposing S phase cells to ionising radiation (IR) induces a transient block to origin firing but does not affect fork rate or fork stalling. Exposure to alkylating agents or UV light causes a slowing of fork movement and a high rate of fork stalling in addition to a sustained block to origin firing. Nucleotide depletion also reduces fork rate, increases stalling and suppresses new origin firing. The block to new origin firing depends on the central checkpoint kinases ATM and ATR in response to damage by IR and UV respectively. Both responses are transduced jointly by the CHK1 and CHK2 kinases. ATR also has a role in preventing irreversible fork stalling but this appears to be independent of CHK1. Finally, the slowing of replication forks is independent of both ATR and CHK1. Thus, this work provides a detailed picture of the mechanics of the replication response to DNA damage in human cells, and clarifies the relative checkpoint dependencies of each aspect of this response.
... The DNA replication process is highly regulated both spatially and temporally, resulting in the changing pattern of replication structures throughout S-phase. The temporal order of DNA replication reflects this higher-order organization of the nuclear genome [1][2][3]. Eu-and heterochromatin, as major higher-order chromatin structures, are defined by a complex interplay between their condensation state, chromatin modifications, associated proteins, as well as their transcriptional activity, all referred to as epigenetic marks [4][5][6]. These epigenetic properties of chromatin regions are potential determinants of their DNA replication timing [7][8][9][10]. ...
... of Experimental Biology, University of Jaén, Jaén, Spain. 3 Institute of Toxicology, Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany. 4 Present Address: Chromosome Replication Laboratory, The Francis Crick Institute, London NW1 1AT, UK. ...
Background
The genome of some vole rodents exhibit large blocks of heterochromatin coupled to their sex chromosomes. The DNA composition and transcriptional activity of these heterochromatin blocks have been studied, but little is known about their DNA replication dynamics and epigenetic composition.
Results
Here, we show prominent epigenetic marks of the heterochromatic blocks in the giant sex chromosomes of female Microtus cabrerae cells. While the X chromosomes are hypoacetylated and cytosine hypomethylated, they are either enriched for macroH2A and H3K27me3 typical for facultative heterochromatin or for H3K9me3 and HP1 beta typical for constitutive heterochromatin. Using pulse-chase replication labeling and time-lapse microscopy, we found that the heterochromatic block enriched for macroH2A/H3K27me3 of the X chromosome is replicated during mid-S-phase, prior to the heterochromatic block enriched for H3K9me3/HP1 beta, which is replicated during late S-phase. To test whether histone acetylation level regulates its replication dynamics, we induced either global hyperacetylation by pharmacological inhibition or by targeting a histone acetyltransferase to the heterochromatic region of the X chromosomes. Our data reveal that histone acetylation level affects DNA replication dynamics of the sex chromosomes’ heterochromatin and leads to a global reduction in replication fork rate genome wide.
Conclusions
In conclusion, we mapped major epigenetic modifications controlling the structure of the sex chromosome-associated heterochromatin and demonstrated the occurrence of differences in the molecular mechanisms controlling the replication timing of the heterochromatic blocks at the sex chromosomes in female Microtus cabrerae cells. Furthermore, we highlighted a conserved role of histone acetylation level on replication dynamics across mammalian species.
... It has long been known that transcriptionally active open euchromatin replicates in early S phase, whereas the structurally compact heterochromatin replicates late (with some exceptions). Microscopically, early replication becomes visible in mammalian cells as hundreds of foci of active replication scattered throughout the nucleus after pulse-labelling with detectable nucleotide analogues or when expressing labelled PCNA (proliferating cell nuclear antigen) [17,[27][28][29][30]. Mid and late S phase replication sites are localised close to heterochromatin, at the nuclear periphery and around nucleoli. ...
... A specific replication timing program was proposed based on the observation that the same replication foci patterns (specific for early, mid and late S phase) re-occurred in every S phase [17,27,28,30]. Elegant nuclei transfer experiments then drew a link between chromatin domain formation and replication timing [28]. ...
Complete genome duplication is essential for genetic homeostasis over successive cell generations. Higher eukaryotes possess a complex genome replication program that involves replicating the genome in units of individual chromatin domains with a reproducible order or timing. Two types of replication origin firing regulations ensure complete and well-timed domain-wise genome replication: (1) the timing of origin firing within a domain must be determined and (2) enough origins must fire with appropriate positioning in a short time window to avoid inter-origin gaps too large to be fully copied. Fundamental principles of eukaryotic origin firing are known. We here discuss advances in understanding the regulation of origin firing to control firing time. Work with yeasts suggests that eukaryotes utilise distinct molecular pathways to determine firing time of distinct sets of origins, depending on the specific requirements of the genomic regions to be replicated. Although the exact nature of the timing control processes varies between eukaryotes, conserved aspects exist: (1) the first step of origin firing, pre-initiation complex (pre-IC formation), is the regulated step, (2) many regulation pathways control the firing kinase Dbf4-dependent kinase, (3) Rif1 is a conserved mediator of late origin firing and (4) competition between origins for limiting firing factors contributes to firing timing. Characterization of the molecular timing control pathways will enable us to manipulate them to address the biological role of replication timing, for example, in cell differentiation and genome instability.
... They also enabled researchers to collect a new data about the organization of replication. In this respect, it was, for example, proven that during the S phase, three basic replication patterns can be observed (Figure 3), or if described in more detail, five patterns can be observed in mammalian cells [64][65][66]. The double labeling of replicating DNA is based on the use of two different clones of primary antibodies raised against BrdU. ...
... They also enabled researchers to collect a new data about the organization of replication. In this respect, it was, for example, proven that during the S phase, three basic replication patterns can be observed (Figure 3), or if described in more detail, five patterns can be observed in mammalian cells [64][65][66]. ...
The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized. Several methods are used for their detection, including the highly popular click chemistry. This review aims to provide the readers with basic information about the various possibilities of the detection of replication activity using nucleoside and nucleotide analogues, and to show the strengths and weaknesses of those different detection systems, including click chemistry for microscopic studies.
... Early studies in mammalian cells have shown that, during S phase, replication takes place at discrete replication foci within the nucleus [221]. Such replication foci have later been referred to as replication factories, since several replisomes act together within these foci to duplicate the genome in a synchronous manner [222,223]. ...
In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive 'identity code' that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.
... During S phase progression, the number and localization of these foci change due to the coordination of origin firing at distinct chromatin and nuclear regions ( Figure 1G) (41). Abnormal patterns of DNA synthesis can be observed in cells with deficient spatiotemporal control of DNA replication ( Figure 1H), as described in the absence of factors important for DNA replication timing control, such as Treslin or RIF1 (42)(43)(44)(45)(46)(47). Interestingly, cells deficient for ATXN3 displayed an increase in abnormal EdU DNA replication patterns ( Figure 1I), in addition to significant differences in some of the regular replication patterns (Supplementary Figure S1J). ...
The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.
... Microscopic analysis of the spatial distribution of replication foci during S-phase identified a set of specific patterns. These patterns appear in a rather strict temporal order during the genome replication with early patterns colocalizing with euchromatin while late patterns are similar to the distribution of heterochromatin (Nakamura et al., 1986;Manders et al., 1996;Zink et al., 1999). Tight correlation of the replication timing and replication patterns with transcriptional state was further confirmed by molecular approaches (Pope et al., 2013;Zhao et al., 2020). ...
A detailed understanding of the principles of the structural organization of genetic material is of great importance for elucidating the mechanisms of differential regulation of genes in development. Modern ideas about the spatial organization of the genome are based on a microscopic analysis of chromatin structure and molecular data on DNA–DNA contact analysis using Chromatin conformation capture (3C) technology, ranging from the “polymer melt” model to a hierarchical folding concept. Heterogeneity of chromatin structure depending on its functional state and cell cycle progression brings another layer of complexity to the interpretation of structural data and requires selective labeling of various transcriptional states under nondestructive conditions. Here, we use a modified approach for replication timing-based metabolic labeling of transcriptionally active chromatin for ultrastructural analysis. The method allows pre-embedding labeling of optimally structurally preserved chromatin, thus making it compatible with various 3D-TEM techniques including electron tomography. By using variable pulse duration, we demonstrate that euchromatic genomic regions adopt a fiber-like higher-order structure of about 200 nm in diameter (chromonema), thus providing support for a hierarchical folding model of chromatin organization as well as the idea of transcription and replication occurring on a highly structured chromatin template.
... Historically, the first evidence for a spatiotemporally conserved compartmentalization of DNA replication stems from pulse-labeling and immunofluorescence microscopy experiments with nucleoside analoga (Nakamura et al., 1986). Punctuate sites of replication were described ("replication foci", RFi) that encompass distinct spatial compartments during early, mid and late S phase and re-appear in S phases of daughter cells (Jackson and Pombo, 1998). ...
... DNA replication timing is independent of the spatial distribution of replication foci. DNA replication takes place in a spatially organised manner 54,55 , with the distribution of replication foci correlated to the time of replication 56 . We have shown that in mouse primary embryonic fibroblasts (pMEFs), Rif1 deficiency induces changes of both the spatial distribution of replication foci and replication timing 12 . ...
Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.
... However, this pattern is conserved throughout several rounds of replication within the same cell type. This suggested that replication foci are a fundamental structure of mammalian chromosomes (Ma et al., 1998a;Nakamura et al., 1986a). ...
Why solid tumours show high chromosome instability is still poorly understood. Seminal work in the host lab has shown in the yeast model system that precocious CDK activation and reduced origin licensing in G1 cause S-phase extension and chromosome rearrangements in mitosis. Since most cancer cells have genetic or epigenetic alterations in one or more G1/S cell cycle regulators that can impede origin licensing, we analysed chromosome replication dynamics in fourteen human epithelial cancer cell lines using newly developed techniques, and compared it to normal human fibroblasts, mammary and retinal pigment epithelial cells. Our results show that all cancer cells spend longer time in S phase (10-29h) than untransformed cells (7-9h). Interestingly, most cancer cell lines displayed a lower global instant density of replication forks (GIFD), partly compensated for some cell lines by increased fork velocity (FV). We define replication potency (RP = GIFD x FV) as a new descriptor of cells’ capacity to synthesize DNA that integrates this compensation mechanism, and found that it was lower for cancer cell lines.The consequences of this longer S phase on the cell cycle and mitosis were assessed by 4D microscopy. We detected mitotic DNA synthesis (MiDAS) and chromosome segregation failures in cancer cells not treated with replication drugs, indicating constitutive chromosome instability (CIN). Importantly, the low GIFD and long S phase of pRb+ cancer cells was reversed by slightly extending G1 using low dose of the CDK4/6 inhibitor Palbociclib. Our data strongly suggest that S-phase extension due to lowered origin licensing in G1 is a common feature and perhaps the main trigger for the genomic instability in cancer cells.
... Long before the development of high-throughput genomic techniques, cytological and imaging techniques served as the major avenue for studying replication. Labeling cells with nucleotide analogs, initially tritiated thymidine and later halogenated nucleotides that could be detected with fluorescent antibodies, identified both a temporal and a spatial regulation of replication within the nucleus (Taylor 1960;Stubblefield 1975;Nakamura et al. 1986). DNA synthesis could be seen to take place in punctate "replication foci" that were localized to the interior of the nucleus in early S phase, moving to the nuclear and nucleolar periphery during late S phase, defining "early and late" spatiotemporal chromatin compartments ( Fig. 2A). ...
Immediately following the discovery of the structure of DNA and the semi-conservative replication of the parental DNA sequence into two new DNA strands, it became apparent that DNA replication is organized in a temporal and spatial fashion during the S phase of the cell cycle, correlated with the large-scale organization of chromatin in the nucleus. After many decades of limited progress, technological advances in genomics, genome engineering, and imaging have finally positioned the field to tackle mechanisms underpinning the temporal and spatial regulation of DNA replication and the causal relationships between DNA replication and other features of large-scale chromosome structure and function. In this review, we discuss these major recent discoveries as well as expectations for the coming decade.
... Early microscopic studies of the replicating genome during Sphase provided a first opportunity to explore its genome-wide partitioning into discrete structural entities with a DNA content of~1 Mb, called RDs or replication foci 58,59 . We adopted the term~1 Mb chromatin domains in line with evidence that RDs persist as similarly sized stable chromatin units throughout interphase and during subsequent cell cycles 33,34 . ...
Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we !nd that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically de!ned, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatiotemporal
patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
... During the replication process, origins fire in many discrete sites that can be visualized by immuno-labelling of thymidine analogues. Different foci pattern exist within S phase nuclei among which we can identify small early replication foci predominantly at the centre of the nucleus and late replicating foci that are bigger and usually found at the nuclear or nucleolar periphery (Nakamura et al., 1986). This three dimensional replication foci pattern is highly conserved in eukaryotes among specifies, as it has been observed in plants and other animals as well (Alexandrova et al., 2003;Mayr et al., 2003;Postberg et al., 2005). ...
DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents.
... A role of IC-channels as additional structural boundaries between CDs and CDCs located on both sides, has been considered but not proven 17 . Early microscopic studies of the replicating genome during Sphase provided a first opportunity to explore its genome-wide partitioning into discrete structural entities with a DNA content of~1 Mb, called RDs or replication foci 58,59 . We adopted the term~1 Mb chromatin domains in line with evidence that RDs persist as similarly sized stable chromatin units throughout interphase and during subsequent cell cycles 33,34 . ...
Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
... Since RIF1 has been recently shown to function in a tissue-specific manner 42 Figure 5B, D). The spatial distribution of these foci evolves in a stereotype fashion during S phase: from numerous small ones located throughout the nucleus in early-S phase, to few large punctuated ones in mid/late-S phase [43][44][45][46] . Our analysis revealed decreased proportion of cells exhibiting a mid-late versus early S-phase patterns in Rif1 morphants. ...
Stemness could be defined as a state in which a cell is able to self-renew and/or to differentiate after cell division. Before this happens, exhaustive duplication of the genome free of errors must occur in order to avoid deleterious mutations, a hallmark of cancer. Thus, DNA replication is particularly important to stem cells because of their continuous division capacities. Regarding DNA replication in eukaryotes, it was discovered that segments of chromosomes close in space, replicate in a coordinated manner during S phase, a process called replication timing. Moreover, major changes in replication timing correlate with cell differentiation, 3D chromatin architecture and transcription. However, the molecules that govern its regulation are poorly understood. Previously, my laboratory found that YAP, the downstream effector of the Hippo pathway, regulates S phase progression of retinal stem cells in Xenopus laevis. To test YAP function in the direct control of replication timing, we took advantage of the powerful in vitro DNA replication system of X. laevis egg extracts. Briefly, we discovered that YAP is recruited to replicating chromatin dependently of origin licensing. In addition, YAP depleted extracts showed increased DNA synthesis and origin activation; revealing that YAP normal function is to slow-down replication by limiting origin firing. Interestingly, we found Rif1, a major regulator of replication timing, as a novel partner of YAP. In vivo, Rif1 expression overlaps that of Yap within the stem cell compartment of the Xenopus retina. Knockdown of Rif1 leaded to a small-eye phenotype and alterations in replication foci of retinal stem cells, resembling the effect observed in YAP deficient cells. Finally, early-embryonic depletion of both molecules resulted in a strikingly acceleration of cell division.Altogether, our findings unveil YAP implication in the regulation of replication dynamis and show Rif1 as a novel partner. Further investigation to analyze this interaction would help us to understand the biological relevance in the control of replication timing and whether it could be used as a target in regenerative medicine.
... Initial studies of chromatin organization revealed the presence of 1 megabase wide focal DNA replication structures that were spatially stable over several cell cycles and were thought to represent chromatin structures (Nakamura et al., 1986;Nakayasu and Berezney, 1989). It was later shown that these regions contained aggregates of several 100kb domains/loops (Jackson and Pombo, 1998, Ma et al., 1998, Berezney and Wei, 1998, Zink et al., 1999, Cremer et al., 2000. ...
Our genome is constantly under attack by endogenous and exogenous factors which challenge its integrity and lead to different types of damages. Double strand breaks (DSBs) constitute the most deleterious type of damage since they maylead to loss of genetic information, translocations and cell death. All the repair processes happen in the context of a highly organized and compartmentalized chromatin. Chromatin can be divided into an open transcriptionally active compartment (euchromatin) and a compacted transcriptionally inactive compartment (heterochromatin). These different degrees of compaction play important roles in regulating the DNA damage response. The goal of my first project was to understand the influence of 3D genome organization on DNA repair. I used two complementary approaches to induce and map DSBs in the mouse genome. My results have shown that enrichment of the DNA damage repair factor γH2AX occurs at distinct loci in the mouse embryonic stem cell genome and that the damage persists in the heterochromatin compartment while the euchromatin compartment is protected from DNA damage. For my second project, I mapped the genomic footprint of 53BP1, a factor involved in DSBs repair, in asynchronous and G1 arrested U2OS cells to identify novel 53BP1 binding sites. My results have identified novel 53BP1 binding domains which cover broad regions of the genome and occur in mid to late replicating regions of the genome.
... Many observations of the incorporation of nucleotide analogues during S phase suggest that replication occurs at discrete foci within nuclei w hich may represent the association of m any replication forks (Cox and Laskey, 1991;Leno and Laskey, 1991;Mills et al., 1989;Nakam ura et al., 1986;Nakayasu and Berezney, 1989; N ew port and Yan, 1996;Pasero et al., 1997). Several replication proteins including RPA, PCNA and polymerase a, cyclin A and Cdk2 co-localise w ith sites of DNA replication in the nucleus (Bravo and Macdonald-Bravo, 1987) (Adachi and Laemmli, 1992;Cardoso et al., 1993;Hozak et al., 1993). ...
DNA replication must occur once and only once in every cell cycle to ensure that mitosis produces two daughter cells with the same complement of genomic DNA. Initiation of DNA replication depends upon pre-replicative complex (pre-RC) assembly at origins during Gl. The pre-RC includes a six-subunit origin recognition complex (ORC) and Cdc6p which together load a complex of the Mcm2-7p family of putative helicases onto chromatin. Only one ORC subunit, Orc6p, is not required for binding of the others to origins in vitro. Using a temperature sensitive orc6 mutant we have shown in vivo that although Orc6p is dispensable for binding of other ORC subunits to chromatin, it is essential for pre-RC formation and may function as an "adapter" between ORC and other pre-RC components. The activity of cyclin dependent kinases (CDKs) regulates pre-RC formation in the cell cycle. CDKs inhibit the formation of pre-RCs during S phase, G2 and M phase, suggesting that inactivation of CDKs at the end of mitosis is essential for pre- RC formation in Gl. Other mitotic events may also be important for pre-RC formation. In particular, the anaphase promoting complex (APC/C), which targets mitotic proteins for proteolysis and the mitotic exit network which activates a mitotic phosphatase, Cdcl4p, have been implicated in pre-RC formation. We have found that inactivation of CDKs during mitosis bypasses the requirement for the APC/C and the mitotic exit network in DNA replication. This suggests that the only essential role for these proteins for pre-RC formation is to bring about CDK inactivation at the end of mitosis.
... A variety of subsequent studies [71][72][73] using nucleotide pulse labeling and microscopical analysis established the existence of functional units of DNA replication in different rodent and marsupial cell lines and, furthermore, described the focal pattern changes throughout S-phase. ...
Decades of investigation on genomic DNA have brought us deeper insights into its organization within the nucleus and its metabolic mechanisms. This was fueled by the parallel development of experimental techniques and has stimulated model building to simulate genome conformation in agreement with the experimental data. Here, we will discuss our recent discoveries on the chromatin units of DNA replication and DNA damage response. We will highlight their remarkable structural similarities and how both revealed themselves as clusters of nanofocal structures each on the hundred thousand base pair size range corresponding well with chromatin loop sizes. We propose that the function of these two global genomic processes is determined by the loop level organization of chromatin structure with structure dictating function.
Abbreviations: 3D-SIM: 3D-structured illumination microscopy; 3C: chromosome conformation capture; DDR: DNA damage response; FISH: fluorescent in situ hybridization; Hi-C: high conformation capture; HiP-HoP: highly predictive heteromorphic polymer model; IOD: inter-origin distance; LAD: lamina associated domain; STED: stimulated emission depletion microscopy; STORM: stochastic optical reconstruction microscopy; SBS: strings and binders switch model; TAD: topologically associated domain
... For instance, it is estimated in [7] that the length of the replicon (loop) is about 100Kbp. Then the number of full-twists in one replicon (loop) is given by: ...
A double strand DNA has a double helical structure and it is modeled by a thin long twisted ribbon fixed at the both ends. A DNA-link is a topological model of such a DNA segment in the nuclear of a eukaryotic cell. In the cell cycle, the DNA is replicated and distributed into new cells. The complicated replication process follows the semi-conservative scheme in which each backbone string is preserved in the replicated DNA. This is interpreted in terms of splitting process of the DNA-link. In order to split the DNA-link, unknotting operations are required. This paper presents a recursive unknotting operations, which efficiently reduce the number of twistings.
... We performed a pulse with 5-bromo-2-deoxyuridine (BrdU) for 2 hours 230 ( Figure 5A) before carrying out BrdU immunofluorescence at telomeres in interphase cells ( Figure 231 5B-C). Only non S-phase cells were counted in this experiment, based on the formation of clear 232 BrdU foci (Dilley et al., 2016;Nakamura, Morita, & Sato, 1986) (Figure 5B). TRF1 -/-MEFs display 233 elevated BrdU incorporation at telomeres, showing two-times more telomere synthesis compared to 234 control cells ( Figure 5C). ...
Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.
... For instance, it is estimated in [7] that the length of the replicon (loop) is about 100Kbp. Then the number of full-twists in one replicon (loop) is given by: 100Kbp 10.5bp ≈ 9.5K. ...
A double strand DNA has a double helical structure and it is modeled by a thin long twisted ribbon fixed at the both ends. A DNA-link is a topological model of such a DNA segment in the nuclear of a eukaryotic cell. In the cell cycle, the DNA is replicated and distributed into new cells. The complicated replication process follows the semi-conservative scheme in which each backbone string is preserved in the replicated DNA. This is interpreted in terms of splitting process of the DNA-link. In order to split the DNA-link, unknotting operations are required. This paper presents a recursive unknotting operations, which efficiently reduce the number of twistings.
... We performed a pulse with 5-bromo-2-deoxyuridine (BrdU) for 2 hours 186 ( Figure 4A) before carrying out BrdU immunofluorescence at telomeres in interphase cells ( Figure 187 4B-C). Only non S-phase cells were counted in this experiment, based on the formation of clear BrdU 188 foci ( Dilley et al., 2016;Nakamura, Morita, & Sato, 1986) (Figure 4B). TRF1 -/-MEFs display 189 elevated BrdU incorporation at telomeres, showing eight times more telomere synthesis (positive 190 cells with more than 5 foci) compared to control cells ( Figure 4C). ...
Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that telomeres lacking TRF1 undergo protein composition reorganisation associated with a DNA damage response and chromatin remodelers. Surprisingly, TRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for TRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.
... DNA synthesis is observed in discrete replication foci identified by replisome components and pulses of labelled nucleotides [16]. The spatiotemporal pattern of replication foci is consistent from one cell cycle to the next and identifies early, middle and late S phase [17,18]. ...
Mechanisms that limit origin firing are essential as the ˜50,000 origins that replicate the human genome in unperturbed cells are chosen from an excess of ˜500,000 licensed origins. Computational models of the spatiotemporal pattern of replication foci assume that origins fire stochastically with a domino-like progression that places later firing origins near recent fired origins. These stochastic models of origin firing require dormant origin signaling that inhibits origin firing and suppresses licensed origins for passive replication at a distance of ∼7–120 kbp around replication forks. ATR and CHK1 kinase inhibitors increase origin firing and increase origin density in unperturbed cells. Thus, basal ATR and CHK1 kinase-dependent dormant origin signaling inhibits origin firing and there appear to be two thresholds of ATR kinase signaling. A minority of ATR molecules are activated for ATR and CHK1 kinase-dependent dormant origin signaling and this is essential for DNA replication in unperturbed cells. A majority of ATR molecules are activated for ATR and CHK1 kinase-dependent checkpoint signaling in cells treated with DNA damaging agents that target replication forks. Since, ATR and CHK1 kinase inhibitors increase origin firing and this is associated with fork stalling and extensive regions of single-stranded DNA they are DNA damaging agents. Accordingly, the sequence of administration of ATR and CHK1 kinase inhibitors and DNA damaging agents may impact the DNA damage induced by the combination and the efficacy of cell killing by the combination.
... This was later supported by replication banding studies [17] and subsequently by microscopic observations of replicated DNA [42]. That is, since the 1980s a number of groups have carried out microscopic experiments in which replicated sequences were labeled with nucleotide analogs and visualized in the nucleus by immunofluorescence using antibodies specific to these nucleotide analogs [42][43][44][45][46]. As a result, it was concluded that each stretch of DNA replicated within~60 min could be observed as a discrete bright spot in the nucleus that was named the replication focus. ...
In mammalian cells, DNA replication timing is controlled at the level of megabase (Mb)-sized chromosomal domains and correlates well with transcription, chromatin structure, and three-dimensional (3D) genome organization. Because of these properties, DNA replication timing is an excellent entry point to explore genome regulation at various levels and a variety of studies have been carried out over the years. However, DNA replication timing studies traditionally required at least tens of thousands of cells, and it was unclear whether the replication domains detected by cell population analyses were preserved at the single-cell level. Recently, single-cell DNA replication profiling methods became available, which revealed that the Mb-sized replication domains detected by cell population analyses were actually well preserved in individual cells. In this article, we provide a brief overview of our current knowledge on DNA replication timing regulation in mammals based on cell population studies, outline the findings from single-cell DNA replication profiling, and discuss future directions and challenges.
... doi: bioRxiv preprint first posted online Feb. 1, 2019; interactions may be driven by cooperative interactions between Fkh1--bound origins recruiting limiting initiation factors such as Dbf4, Sld3 and Cdc45. This aggregation of origins selected for early/efficient activation has the inevitable consequence that replication initiation transforms these origin clusters into replication foci, which have been observed as concentrations of DNA synthesis and replication factors (Berezney et al., 2000;Frouin et al., 2003;Hozak et al., 1994;Kitamura et al., 2006;Nakamura et al., 1986;Newport and Yan, 1996). These assemblages may contribute to efficient chromosomal replication initiation and elongation in multiple ways, such as accretion of activities and co--factors directly required for DNA synthesis (e.g.: dNTP production), and scaffolding to co--localize and coordinate replication with related activities like chromatin assembly, cohesion establishment, topological resolution, and DNA repair. ...
Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. Sites undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In S. cerevisiae , replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase. Here, we link localization of replication origins in G1 phase with Fkh1 activity, which is required for their early replication timing. Using a Fkh1-dependent origin relocalization assay, we determine that execution of Dbf4-dependent kinase function, including Cdc45 loading, results in dynamic relocalization of a replication origin from the nuclear periphery to the interior in G1 phase. Origin mobility increases substantially with Fkh1-driven relocalization. These findings provide novel molecular insight into the mechanisms that govern dynamics and spatial organization of DNA replication origins and possibly other functional DNA elements.
... In mammalian cells, replication foci characteristic of actively replicating chromosomal regions are localized to the interior of the nucleus in early S phase, while they are localized to the nuclear periphery from mid to late S phase (Nakamura et al, 1986;Nakayasu & Berezney, 1989;O'Keefe et al, 1992). Prior studies suggest that a spatiotemporal replication program is established at a specific time point (timing decision point, TDP) in the G1 phase (Dimitrova & Gilbert, 1999). ...
DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.
... The duplication of the genome is a highly complex process organized in a spatial and temporal manner (reviewed in (1)). On a cytological level, DNA replication is detectable as discrete sub-nuclear foci, where each focus corresponds to a cluster of coordinately activated replication forks (2)(3)(4)(5), which can be resolved using superresolution light microscopy (6)(7)(8). During S-phase progression, the spatial distribution of these foci changes 'following' chromatin condensation level and leading to distinct nuclear patterns associated with early (euchromatin), mid (facultative heterochromatin) and late replicating (constitutive heterochromatin) chromosomal regions ( Figure 1). ...
The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.
... A large body of evidence exists that DNA replication occurs in the replisomes located on the nuclear matrix and that halo loops correspond to active and probably dormant replicons, with replication origins located at the base of loops (Vaughn et al. 1990;Anachkova et al. 2005). During replication, looped DNA passes through replisomes, where the DNA is unwound with topoisomerase (Nakamura et al. 1986;Razin 2001;Anachkova et al. 2005;Rivera-Mulia et al. 2011). Most loops of interphase telomeric halos in humans and other mammalian species probably appear as single replicons, with the replication origin located in the subtelomeric region. ...
Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ɤ-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.
... These compartments gather together DNA polymerase and replication cofactors where the DNA duplication of multiple DNA strands proceeds at the same time (Nakamura et al., 1986;Jackson and Pombo, 1998). An alternative way to investigate the spatial organization of the replication program came with the advent of the chromosome conformation capture (3C) technique (Dekker et al., 2002;Lieberman-Aiden et al., 2009). ...
Decades of studies showed that chromatin structure is tightly linked to DNA related metabolic processes, through the dynamic regulation of a myriad of molecular factors. The proper organization of chromosomes is notably important to ensure the maintenance of DNA integrity during cell cycle progression. Using the model S. cerevisiae, the aim of my PhD project was to characterize to which extent chromatin reorganization during the cell cycle may influence chromosome stability. To do so, we first generated a comprehensive genome-wide study of the reorganization of yeast’s chromosomes during an entire cell cycle. This work, besides recapitulating expected chromosomal features of the replication and mitotic stages, led to the characterization of peculiar chromosome structures such as a DNA loop bridging the rDNA and the centromeres. The role of structural maintenance of chromosomes (SMC) complexes and of microtubules were thoroughly investigated. A second part of my work focused on describing features of the chromatin organization of cells that exited the proliferative cell cycle and entered into quiescence. We characterized the dense status of silenced heterochromatin at specific loci, such as telomeres, in relation to the silent information regulators (SIRs). Finally, we tried to achieve a better understanding of the functional interplay between chromosome stability and the 3D genome architecture during replication, by investigating the genomic stability at replication pausing sites. Overall, our results point at a striking plasticity of replication structures to different stresses. Future work aims to map replication-dependent chromosomal rearrangements on the genomic maps.
... Duplication of the genome occurs in a defined temporal order known as the replication timing (RT) programme (Rhind and Gilbert 2013). Early cytogenetic studies of DNA replication that visualized DNA synthesis by incubating S phase mammalian cells with nucleotide homologues such as BrdU revealed distinct punctate structures in the nucleus termed replication foci that were found in different spatial patterns at distinct times during S phase (Nakamura et al. 1986;Nakayasu and Berezney 1989). Early in S phase, many small replication foci are associated with euchromatic regions interspersed throughout the nucleoplasm, while as S phase progresses, DNA synthesis moves to the heterochromatin-rich regions at nuclear and nucleolar peripheries forming what appeared under conventional light microscopy to be larger foci. ...
DNA replication occurs in a defined temporal order during S phase, known as the replication timing programme, which is regulated not only during the cell cycle but also during the process of development and differentiation. The units of replication timing regulation, known as replication domains (RDs), frequently comprise several nearly synchronously firing replication origins. Replication domains correspond to topologically associating domains (TADs) mapped by chromatin conformation capture methods and are likely to be the molecular equivalents of replication foci observed using cytogenetic methods. Both TAD and replication foci are considered to be stable structural units of chromosomes, conserved through the cell cycle and development, and accordingly, the boundaries of RDs also appear to be stable in different cell types. During both normal development and progression of disease, distinct cell states are characterized by unique replication timing signatures, with approximately half of genomic RDs switching replication timing between these cell states. Advances in functional genomics provide hope that we can soon gain an understanding of the cause and consequence of the replication timing programme and its myriad correlations with chromatin context and gene regulation.
Background Factor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C type lectin domain
(CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector.
Methods and results The FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted
by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h.
Conclusions Protein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression.
We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating
Background : It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented.
Methods : We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome.
Results : The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation order observed by microscopy.
Conclusions : We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.
Background : It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the location of origin clusters on the genome has not been documented.
Methods : We synchronised human U2OS by thymidine block and release followed by a brief block with L-mimosine to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped back onto the human genome.
Results : The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters were preferentially activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation observed by microscopy.
Conclusions : We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.
DNA double-strand breaks (DSBs) are lesions that arise frequently from exposure to damaging agents as well as from ongoing physiological DNA transactions. Mis-repair of DSBs leads to rearrangements and structural variations in chromosomes, including insertions, deletions, and translocations implicated in disease. The DNA damage response (DDR) limits pathologic mutations and large-scale chromosome rearrangements. DSB repair initiates in 2D at DNA lesions with the stepwise recruitment of repair proteins and local chromatin remodeling which facilitates break accessibility. More complex structures are then formed via protein assembly into nanodomains and via genome folding into chromatin loops. Subsequently, 3D reorganization of DSBs is guided by clustering forces which drive the assembly of repair domains harboring multiple lesions. These domains are further stabilized and insulated into condensates via liquid-liquid phase-separation. Here, we discuss the benefits and risks associated with this 3D reorganization of the broken genome.
A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a “cloudy nebula” to numerous numbers of “beads on a string” and finally to “striped arrays of banding structured chromosome” known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.Key wordsChromosome condensation/compactionChromosome structure
DNA replication
Replication foci
Premature chromosome condensation (PCC)
Prematurely condensed chromosomes (PCCs)
Calyculin A
Chromosome territories
Beads loading method
Cy3-dUTP
Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.
Immunofluorescence techniques have been used to investigate the distributions of a number of host cell proteins upon infection with three different DNA tumour viruses: herpes simplex virus type 1 (HSV-1), adenovirus (types 2, 4 and 5) and simian virus 40 (SV40). A number of host DNA replication proteins were shown to specifically redistribute to viral replication "compartments" labelled by antibody to the HSV-1 major DNA-binding protein (ICP8) during productive HSV-1 infection. These host proteins also colocalised to varying extents with ICP8 in the presence of a specific inhibitor of viral DNA replication which caused a marked alteration in the location of intranuclear ICPB. Two anti- oncogenic proteins, retinoblastoma and p53 were shown to relocate in the same manner as the cellular DNA replication proteins during both productive and abortive HSV-1 infections, raising the possibility that these proteins may be associated with DNA replication complexes in uninfected cells. Transfection experiments showed that the seven HSV-1 proteins shown to be essential for DNA replication can induce replication compartment formation in uninfected cells. Host DNA replication proteins were also redistributed during productive (but not abortive) infection with adenovirus, although, of those tested, only proliferating cell nuclear antigen (PCNA) was detected in viral replication compartments. A specific population of PCNA was also detected at novel discrete nuclear foci. SV40 infection resulted in the redistribution of singlestranded DNA-binding protein (SSB) to foci which enlarged as infection progressed and also contained SV40 large T antigen. SV40 infection of adenovirus-transformed 293 cells caused p53 to move from the cytoplasm to the nucleus. SV40 co-infections with adenovirus and HSV-1 were also studied. The production and characterisation of monoclonal antibodies to the HSV-1 major DNA binding protein (ICP8) and polymerase accessory factor (UL42) are also described.
The eukaryotic cytoplasm is a complex, organized and highly dynamic environment, whose dynamic organization is dependent on a network of filaments and associated proteins. These networks are established, accepted and well studied. The nucleus is no less complex, organized or dynamic, but the existence of an equivalent nuclear network in is not universally accepted and has proved difficult to study. Theoretically there seems an almost overwhelming requirement for a nucleoskeleton, which is needed to provide a structural framework to organize the genome, as well as other subnuclear components, into functionally distinct regions. Such organization must be, and is, highly dynamic so that it can change during development and in response to changing requirements of the cell. Unfortunately, filamentous structures in the nucleus are difficult to detect amongst all the other fibrous material (the chromatin), which has to be removed in order to visualize the putative nucleoskeleton. Therefore, although such structures can be prepared from both plants and animals, their in vivo relevance has remained contentious. Nucleoskeletal filaments have the appearance of intermediate filaments and in fact animal cells have a clearly defined intermediate filament network at the nuclear periphery: the nuclear lamina. Plant cells, however, have no proteins that are clear equivalents of the lamins, or indeed any other intermediate filament protein. In this review we discuss the evidence for nuclear intermediate filament‐like proteins and other potential nucleoskeleton components in plants and discuss their possible roles in plant nuclear organization.
This chapter discusses transcription by RNA polymerase II and nuclear architecture. Nuclear transcription is carried out by three different DNA-dependent RNA polymerases, termed as RNA polymerase I, II, and III (RPI, RPII, RPIII). It is found that anti-RPII antibodies may not discriminate between RPII molecules that are engaged in transcription and those that are inactive. It is suggested that the RPII-antigen distribution may be an overestimation of the number of RPII transcription sites. It is observed that antibodies directed against DNA–RNA hybrids label specific structures in the nucleoplasm and the nucleolus. These structures correspond largely to structures that become labeled by [3H]uridine. It is observed that RPII transcription sites are distributed throughout the nucleus, without any preference for either the nuclear interior or the periphery. It is found that actively transcribed genes are located predominantly at the periphery of domains of condensed chromatin. It is suggested that intron- and exon-specific probes can be used to study the localization of successive steps in RNA processing and transport of pre-mRNA and mature RNA from the site of synthesis to the nuclear envelope.
The human genome forms thousands of "contact domains", which are intervals of enhanced contact frequency. Some, called "loop domains" are thought to form by cohesin-mediated loop extrusion. Others, called "compartmental domains", form due to the segregation of active and inactive chromatin into A and B compartments. Recently, Hi-C studies revealed that the depletion of cohesin leads to the disappearance of all loop domains within a few hours, but strengthens compartment structure. Here, we combine live cell microscopy, super-resolution microscopy, Hi-C, and studies of replication timing to examine the longer-term consequences of cohesin degradation in HCT-116 human colorectal carcinoma cells, tracking cells for up to 30 hours. Surprisingly, cohesin depleted cells proceed through an aberrant mitosis, yielding a single postmitotic cell with a multilobulated nucleus. Hi-C reveals the continued disappearance of loop domains, whereas A and B compartments are maintained. In line with Hi-C, microscopic observations demonstrate the reconstitution of chromosome territories and chromatin domains. An interchromatin channel system (IC) expands between chromatin domain clusters and carries splicing speckles. The IC is lined by active chromatin enriched for RNA Pol II and depleted in H3K27me3. Moreover, the cells exhibit typical early-, mid-, and late- DNA replication timing patterns. Our observations indicate that the functional nuclear compartmentalization can be maintained in cohesin depleted pre- and postmitotic cells. However, we find that replication foci - sites of active DNA synthesis - become physically larger consistent with a model where cohesin dependent loop extrusion tends to compact intervals of replicating chromatin, whereas their genomic boundaries are associated with compartmentalization, and do not change.
Acute promyelocytic leukemia (APL) is thought to be caused by the t(15,17) translocation that fuses the PML gene to that of the retinoic acid receptor alpha (RAR alpha) and generates a PML/RAR alpha fusion protein. Yet, paradoxically, APL cells are exquisitely sensitive to retinoic acid (RA), as they terminally differentiate upon RA exposure. In this report, we have examined the expression of PML and PML/RAR alpha in normal and APL cells. By immunofluorescence or immunocytochemistry, we show that PML has a speckled nuclear pattern of expression that contrasts with that of PML/RAR alpha (mostly a micropunctuated nuclear pattern or a cytoplasmic localization). The APL- derived cell line NB4 that expresses both the PML and PML/RAR alpha genes also shows the fine micropunctuated nuclear pattern, suggesting a dominant effect of the fusion protein over the localization of wild- type PML. RA treatment of NB4 cells or clones expressing PML/RAR alpha gradually leads to a PML pattern before apparent morphologic maturation. In 14 untreated APL patients, the PML-reactive proteins were cytoplasmic (by immunocytochemistry) or both cytoplasmic and nuclear with a micropunctuated pattern (by immunofluorescence). Strikingly, in 4 patients, after 1 to 2 weeks of RA therapy, the speckled nuclear PML pattern reappeared concomitant with the onset of differentiation. These results establish that fusion of PML to RAR alpha results in an altered localization of PML that is reverted upon RA treatment. This observation, which highlights the importance of PML, is likely to be a key to unravelling the molecular mechanism of both leukemogenesis and RA-induced differentiation of APL.
The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms. Different genomic regions are replicated at different times during the S phase of the cell cycle, forming early- and late-replicating domains that occupy different locations in the nucleus. The recent identification of specific DNA sequences and long non-coding RNAs that regulate DNA replication timing is providing key insights into the roles of replication timing and into timing and 3D organization.
Chromosomal DNA elements are organized into spatial domains within the eukaryotic nucleus. Sites undergoing DNA replication, high-level transcription, and repair of double-strand breaks coalesce into foci, although the significance and mechanisms giving rise to these dynamic structures are poorly understood. In S. cerevisiae, replication origins occupy characteristic subnuclear localizations that anticipate their initiation timing during S phase. Here, we link localization of replication origins in G1 phase with Fkh1 activity, which is required for their early replication timing. Using a Fkh1-dependent origin relocalization assay, we determine that execution of Dbf4-dependent kinase function, including Cdc45 loading, results in dynamic relocalization of a replication origin from the nuclear periphery to the interior in G1 phase. Origin mobility increases substantially with Fkh1-driven relocalization. These findings provide novel molecular insight into the mechanisms that govern dynamics and spatial organization of DNA replication origins and possibly other functional DNA elements.
Cell nucleus is characterized by strong compartmentalization of structural components in its three-dimensional space. Certain genomic functions are accompanied by changes in the localization of chromatin loci and nuclear bodies. Here we review recent data on the mobility of nuclear components and the role of this mobility in genome functioning.
DNA replication proceeds along spatially and temporally coordinated patterns within the nucleus, thus protecting the genome during the synthesis of new genetic material. While we have been able to visualize replication patterns on DNA fibers for 50 years, recent developments and discoveries have provided a greater insight into how DNA replication is controlled. In this review, we highlight many of these discoveries. Of great interest are the physiological role of the replication timing program, cis and trans-acting factors that modulate replication timing and the effects of chromatin structure on the replication timing program. We also discuss future directions in the study of replication timing.
In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.
Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.
We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.
Recent studies have indicated that the DNA of the eucaryotic nucleus is organized in the form of supercoiled loops. We show here that after depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact (unnicked) loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix. This halo of DNA loops can be reversibly wound using various concentrations of ethidium bromide and is irreversibly unwound when the DNA is gently nicked with DNAase I or exposure to ultraviolet light. These structures, comprising a halo of unwound DNA loops anchored to a nuclear matrix, provided us with a way in which to examine the relationship of DNA replication to the DNA loops. 3T3 cells were pulse-labeled with 3H-thymidine for various periods and autoradiography was performed on the matrix-halo structures. It was found that after a 1 min pulse, >80% of the autoradiographic grains representing newly replicated DNA were found within the nuclear matrix even though the surrounding halo region contained 80% of the total nuclear DNA. With longer pulse times, the autoradiographic grains could be seen to move progressively outward from the nuclear matrix into the halo region. Over 70% of the autoradiographic grains could be chased out of the central matrix into the surrounding halo if the 1 min pulse was followed by a 1 hr incubation with excess unlabeled thymidine. These findings indicate that DNA replication occurs at fixed sites at the base of the loops and suggests that the replicating DNA loops are motile with respect to their nuclear matrix anchorage sites.
In interphase nuclei and metaphase chromosomes, eukaryotic chromosomal DNA is arranged in negatively supercoiled loops whose length averages several tens of kilobases (kb). Supercoiling indicates that the loops are tenaciously bound at both ends through the periodic attachment of DNA to a non-histone protein structural support, termed the nuclear matrix, skeleton or cage in interphase nuclei and the scaffold in metaphase chromosomes1–16. The looped organization has been envisaged to be important in DNA replication2,11,15,17–25 and transcription26–28. We report here a close relationship between average loop length and replicon size in different animal and plant species. Autoradiographic experiments support the hypothesis that DNA synthesis occurs at fixed sites on the nuclear matrix15,20. We also describe a modification of the looped organization occurring during early embryonic development in Xenopus laevis.
The transforming ability in 10% serum medium of the temperature-sensitive mutants of simian virus 40 in the complementation group III (ts640 type mutants) was greatly reduced when the infected rat 3Y1 cells were incubated at the restrictive temperature of 40 degrees or incubated first at 40 degrees for 3 days and then shifted to the permissive temperature of 33 degrees. Transformation did occur efficiently after incubation at 33 degrees or after an initial incubation at 33 degrees for 5 days followed by a shift to 40 degrees. When growth properties of 3Y1 cells transformed at 33 degrees by the group III mutants were examined at 40 degrees, several aspects of the transformed state were rendered temperature-sensitive. These aspects were the ability of cells to grow in low serum (1.5%) medium and to make colonies, in 10% serum medium, on monolayers of untransformed 3Y1 cells and in soft agar. It is concluded that a simian virus 40 gene (cistron III) controls the initiation, as well as at least some aspects of the maintenance, of transformation and that the initiation reaction is a more heat-labile event than the maintenance reaction(s) under the experimental conditions.
Irradiation of synchronized S phase Chinese hamster ovary cells with 313 nm wavelength light inhibited the initiation of replicons in DNA substituted with bromodeoxyuridine but did not affect the replication of unsubstituted DNA occurring simultaneously in the same cells. This result suggests that initiation is inhibited only in the region of the chromosome that sustains damage. Calculations of the frequency of damaged sites suggest that this inhibition, and probably a similar effect induced by X-rays, could be mediated by conformational changes in regions of individual DNA molecules up to several hundred μm in length.
X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.
The localization of newly-replicated DNA in mouse cells of line P815 in culture is studied by high resolution autoradiography. After 20 or 30 sec of incorporation of , the silver grains are found throughout the nucleus with a relatively higher (about two-fold) density over the peripheral region of the nucleus. After a further 1 h of chase with non-radioactive thymidine, or after 19 h of continuous growth with the radioactive precursor, the pattern of the nuclear distribution of label is not appreciably different from that found after a short pulse. Autoradiography, combined with EDTA staining resulting in a preferential bleaching of the chromatin, reveals after a short pulse of that most of the silver grains lie close to, or over, the border zone between condensed chromatin and the interchromatin region. The results are discussed in the context of other recent findings concerning the sites of DNA replication in eukaryotic cells obtained with different cell systems.
Chinese hamster fibroblasts synchronized by metaphase selection, amethopterin or 5-fluorodeoxyuridine were pulsed labelled in early or late stages of DNA synthesis with 3H-thymidine. Label distributions were studied in EM sections and under light microscopy of flattened nuclei. 1.1. Early DNA synthesis occurred in replicons evenly distributed over the nucleus.2.2. Late DNA synthesis was restricted preferentially to replicons on the periphery of the nucleus.3.3. No shift of label occurred during chase periods with unlabelled thymidine.4.4. No significant differences between label distributions at the various stages could be found following different synchronization methods.5.5. The peripheral labelling during late DNA synthesis reflects synthesis in DNA usually present as condensed chromatin on the nuclear membrane.
For determination of whether DNA replication is initiated at the nuclear envelope, synchronized Chinese hamster ovary cells labeled with [(3)H]thymidine were examined by electron microscope radioautography. The cells were synchronized initially by mitotic shake-off and held at the G(1)-S border by 5-fluorodeoxyuridine plus amethopterin. Cells were fixed at 1, 5, 10, and 30 min after the inhibitors were counteracted with [(3)H]thymidine. Radioautographic silver grains in each case were present over the more central parts of nuclei and were generally absent from the region of the nuclear envelope. We conclude that neither initiation nor continuation of DNA replication is associated with the nuclear envelope.
To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.
Monoclonal antibody against microtubule-associated protein-1 produced intranuclear immunofluorescent spots, which disappeared under growth-inhibited conditions caused by serum starvation and saturated cell density in untransformed cells. A change of medium to 10% serum gave rise to the reappearance of nuclear spots before the resumption of DNA synthesis. This reversible change of immunofluorescence was also caused by a temperature shift in rat 3Y1 cells transformed by Simian virus-40-A640 (temperature-sensitive in large T-antigen). The fluorescence decreased during S phase of the cell cycle. In contrast the transformed cells always showed nuclear fluorescence, irrespective of serum concentrations or the cell cycle. Growth-inhibited cells previously treated with detergent and salt revealed nuclear fluorescent spots. This result suggested antigenic modification.
Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase alpha revealed the intranuclear localization of DNA polymerase alpha in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the alpha-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase alpha appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase alpha by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.
We have developed a procedure for simultaneous flow cytometric measurement of cellular DNA content and amount of BrdUrd incorporated into cellular DNA. Propidium iodide was used as a fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescein-labeled goat anti-mouse antibody was used to fluorescently label the bound anti-BrdUrd probe. Bivariate DNA/BrdUrd distributions measured for Chinese hamster ovary cells labeled for 30 min with BrdUrd clearly show the G1-and G2M-phase cells to have low BrdUrd-linked fluorescence and the S-phase cells to have high BrdUrd-linked fluorescence. Cell cycle traverse rates were estimated for Chinese hamster ovary cells from bivariate distributions measured for samples taken periodically after pulse labeling with BrdUrd. Bivariate DNA/BrdUrd distributions were also applied in the analysis of the response of C3H murine bone marrow cells to treatment in vivo with 1-beta-D-arabinofuranosylcytosine (araC). Bivariate distributions were measured for bone marrow cells taken from mice that were pulse labeled with BrdUrd at various times after treatment with araC. The resulting DNA/BrdUrd sequences show the kinetics of recovery from araC and allow discrimination of the araC sterilized cells.
Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication
on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized
with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced
are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured
cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent
staining method and quantitated by flow cytometry.
The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5' ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.
We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with 3H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.
- McCready