PERK inhibits DNA replication during the Unfolded Protein Response via Claspin and Chk1.

Stresses such as hypoxia, nutrient deprivation and acidification disturb protein folding in the endoplasmic reticulum (ER) and activate the Unfolded Protein Response (UPR) to trigger adaptive responses through the effectors, PERK, IRE1 and ATF6. Most of these responses relate to ER homoeostasis; however, here we show that the PERK branch of the UPR also controls DNA replication. Treatment of cells with the non-genotoxic UPR agonist thapsigargin led to a rapid inhibition of DNA synthesis that was attributable to a combination of DNA replication fork slowing and reduced replication origin firing. DNA synthesis inhibition was dependent on the UPR effector PERK and was associated with phosphorylation of the checkpoint adaptor protein Claspin and activation of the Chk1 effector kinase, both of which occurred in the absence of detectable DNA damage. Remarkably, thapsigargin did not inhibit bulk DNA synthesis or activate Chk1 in cells depleted of Claspin, or when Chk1 was depleted or subject to chemical inhibition. In each case thapsigargin-resistant DNA synthesis was due to an increase in replication origin firing that compensated for reduced fork progression. Taken together, our results unveil a new aspect of PERK function and previously unknown roles for Claspin and Chk1 as negative regulators of DNA replication in the absence of genotoxic stress. Because tumour cells proliferate in suboptimal environments, and frequently show evidence of UPR activation, this pathway could modulate the response to DNA replication-targeted chemotherapies.

Molecular combing in the analysis of developmentally regulated amplified segments of Bradysia hygida.

Molecular combing technology is an important new tool for the functional and physical mapping of genome segments. It is designed to identify amplifications, microdeletions, and rearrangements in a DNA sequence and to study the process of DNA replication. This technique has recently been used to identify and analyze the dynamics of replication in amplified domains. In Bradysia hygida, multiple amplification initiation sites are predicted to exist upstream of the BhC4-1 gene. However, it has been impossible to identify them using the available standard techniques. The aim of this study was to optimize molecular combing technology to obtain DNA fibers from the polytene nuclei of the salivary glands of B. hygida to study the dynamics of DNA replication in this organism. Our results suggest that combing this DNA without prior purification of the polytene nuclei is possible. The density, integrity, and linearity of the DNA fibers were analyzed, fibers 50 to 300 kb in length were detected, and a 9-kb fragment within the amplified region was visualized using biotin detected by Alexa Fluor 488-conjugated streptavidin technique. The feasibility of physically mapping these fibers demonstrated in this study suggests that molecular combing may be used to identify the replication origin of the BhC4-1 amplicon.

Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells.

Following prolonged mitotic spindle disruption by microtubule poisons, mammalian cells delay their entry into anaphase, then progressively slip out of mitosis and become tetraploid. Normal cells then stop cycling before S-phase onset, but the mechanisms underlying this arrest are still unclear. Here we show that a double block prevents endo-reduplication. First, cells that exit mitosis without a functional microtubule network are driven toward G0. Reconstitution of the network unmasks a second block that relies on DNA double-strand breaks occurring early in the G1 phase that follows the mitotic block. We propose that a stress signal elicited upon mitotic impairment triggers breakage, which couples the leaky spindle checkpoint to the stringent DNA damage response. Consistent with this finding, cells defective for the damage response continue cycling and acquire, within a single cell cycle, both chromosome rearrangements and abnormal chromosome numbers that remarkably mimic the complex genetic hallmark of tumorigenesis.

Mammalian recombination hot spot in a DNA loop anchorage region: a model for the study of common fragile sites.

We analyzed the replication pattern and the topological organization of a 200 kb long Chinese hamster polygenic locus, which spans the boundary of two isochores. One of them is G + C rich while the second one is highly A + T rich. Previous analysis of mutants amplified for this locus had identified, within the A + T rich isochore, a mitotic recombination hotspot and a replication origin separated by some 7 kb. The recombination hotspot exhibits structural features repeatedly observed at common fragile sites, including a typical enrichment in peaks of enhanced DNA helix flexibility. By studying the replication pattern of the same locus in the non-amplified CHO cells, we confirm here the localization of the replication origin and show that the mitotic recombination hotspot does not correspond to a replicon junction. This finding makes questionable current hypotheses correlating replication termination regions with recombination prone sequences. Using topoisomerase II-mediated DNA cleavage at matrix attachment sites, we identified a 40 kb-long DNA anchorage region extending all along a transcription unit nested within the A + T rich isochore. Both the recombination hotspot and the replication origin lie within this topoisomerase II sensitive region, which suggests that features essential for initiation of recombination and initiation of DNA replication cluster within DNA anchorage regions. Features common to this region and to common fragile sites are discussed. J. Cell. Biochem. Suppl. 36: 170-178, 2001.

Dual control of replication timing. Stochastic onset but programmed completion of mammalian chromosome duplication.

In mammalian cells, DNA replication proceeds according to a precise temporal order during the S phase, but how this program is controlled remains poorly understood. We analyzed the replication-dependent bromodeoxyuridine banding of chromosomes in Chinese hamster cells treated with the spindle poison nocodazole. In these cells, nocodazole induces a transient mitotic arrest, followed by DNA re-replication without intervening cell division. Nuclear fragmentation is often observed in tetraploid derivatives, and previous studies suggest that replication timing of chromosomes could be affected when they are segregated into different micronuclei. Here we show that the onset of replication is frequently asynchronous on individual chromosomes during the re-replication process. Moreover, fluorescence in situ hybridization analysis revealed that replication synchrony is equally altered in fragmented and non-fragmented nuclei, indicating that asynchronous onset of replication is not dependent on physical separation of the chromosomes into isolated compartments. We also show that the ordered program of replication is always preserved along individual chromosomes. Our results demonstrate that the onset of replication of individual chromosomes in the same nuclear compartment can be uncoupled from the time of S-phase entry and from the programmed replication of chromosome sub-domains, revealing that multi-level controls contribute to establish replication timing in mammalian cells.

Enhanced flexibility and aphidicolin-induced DNA breaks near mammalian replication origins: implications for replicon mapping and chromosome fragility.

Common fragile sites are chromosomal loci prone to breakage and rearrangement that can be induced by aphidicolin, an inhibitor of DNA polymerases. Within these loci, sites of preferential DNA breaks were proposed to correlate with peaks of enhanced DNA flexibility, the function of which remains elusive. Here we show that mammalian DNA replication origins are enriched in peaks of enhanced flexibility. This finding suggests that the search for these features may help in the mapping of replication origins, and we present evidence supporting this hypothesis. The association of peaks of flexibility with replication origins also suggests that some origins may associate with minor levels of fragility. As shown here, an increased sensitivity to aphidicolin was found near two mammalian DNA replication origins.

Initiation of DNA replication at the Chinese hamster origin oriGNAI3 relies on local sequences and/or chromatin structures, but not on transcription of the nearby GNAI3 gene.

We recently identified a region of preferential replication initiation, oriGNAI3, near the 3' end of the Chinese hamster GNAI3 gene. oriGNAI3 is co-amplified in mutants selected for AMPD2 amplification, a process generating chromosomal rearrangements. In this report we have taken advantage of cell lines with truncated and translocated amplified units to show that these rearrangements do not alter the function of ori GNAI3. These results indicate that replication initiation at this locus relies essentially on local features. Interestingly, the study of one line in which a rearrangement has disrupted the GNAI3 gene shows that ongoing transcription of this gene is not required for initiation at oriGNAI3. In order to obtain further insight into the sequences and/or chromatin structures required for oriGNAI3 function, we have analyzed the DNase I sensitivity and nucleotide sequence of the region. The features important for replication initiation appear to cluster in a 7-12 kb region which includes oriGNAI3.

A new role for hypoxia in tumor progression: induction of fragile site triggering genomic rearrangements and formation of complex DMs and HSRs.

Genome rearrangements including gene amplification are frequent properties of tumor cells, but how they are related to the tumor microenvironment is unknown. Here, we report direct evidence for a causal relationship between hypoxia, induction of fragile sites, and gene amplification. Recently, we showed that breaks at fragile sites initiate intrachromosomal amplification. We demonstrate here that hypoxia is a potent fragile site inducer and that, like fragile sites inducing drugs, it drives fusion of double minutes (DMs) and their targeted reintegration into chromosomal fragile sites, generating homogeneously staining regions (HSRs). This pathway operates efficiently for DMs bearing different sequences, suggesting a model of hypoxia-driven formation of the HSRs containing nonsyntenic sequences frequently observed in solid tumors.

oriGNAI3: a narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques.

The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes.

Interstitial deletions and intrachromosomal amplification initiated from a double-strand break targeted to a mammalian chromosome.

Interstitial deletions of tumour suppressor genes and amplification of oncogenes are two major manifestations of chromosomal instability in tumour cells. The development of model systems allowing the study of the events triggering these processes is of major clinical importance. Using the properties of the I-SceI nuclease to introduce a localized double-strand break (DSB) in a mammalian chromosome carrying its target sequence, we demonstrate here that both types of mutations can be initiated by non-conservative DSB repair pathways. In our system, I-SceI activity dissociates a transfected gpt gene from its promoter, allowing the isolation of gpt- clones. Our results show that intrachromatid single-strand annealing events occur frequently, giving rise to interstitial deletions not accompanied by other chromosomal rearrangements. We also observed that, when present in the cells, extrachromosomal DNA molecules are integrated preferentially at the broken locus. Taking advantage of the insertion of the I-SceI recognition sequence telomeric to and close to the dihydrofolate reductase gene, we show that a less frequent outcome of I-SceI activity is the initiation of cycles of intrachromosomal amplification of this marker, from breaks at a site merging with the enzyme target.

Gene amplification mechanisms: the role of fragile sites.

We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons.

Drug-selected intrachromosomal gene amplification by breakage-fusion-bridge (BFB) cycles is well documented in mammalian cells, but factors governing this mechanism are not clear. Here, we show that only some clastogenic drugs induce drug resistance through intrachromosomal amplification. We strictly correlate triggering of BFB cycles to induction of fragile site expression. We demonstrate a dual role for fragile sites in intrachromosomal amplification: a site telomeric to the selected gene is involved in initiation, while a centromeric site defines the size and organization of early amplified units. The positions of fragile sites relative to boundaries of amplicons found in human cancers support the hypothesis that fragile sites play a key role in the amplification of at least some oncogenes during tumor progression.

Matrix attachment regions and transcription units in a polygenic mammalian locus overlapping two isochores.

Eukaryotic chromosomes are ponctuated by specialized DNA sequences (MARs) characterized by their ability to bind the network of nonhistone proteins that form the nuclear matrix or scaffold. We previously described an amplifiable cluster of genes with different tissue-specific expression patterns, located on Chinese hamster chromosome 1q. This model is especially appropriate to study the relationships between MARs and transcription units. We show here that four attachment regions, with sequences exhibiting motifs specific to MARs, are present within the 100 kb of screened DNA. Three of them are relatively short sequences localized in intergenic regions. The last one extends over one of the transcription units and contains a region previously identified as a recombination hot spot. Moreover, the analysis of a DNA sequence extending over some 50 Kb of this region and spanning at least four genes, disclosed a strikingly sharp change in G + C content. This strongly suggests that the studied region contains the boundary of two isochores. We propose that the frequency and the size of MARs are correlated to their localization in G + C rich or poor domains.

GNAI3, GNAT2, AMPD2, GSTM are clustered in 120 kb of Chinese hamster chromosome 1q.

We studied a polygenic region located on Chromosome (Chr) 1q in Chinese hamster cells that is coamplified along with the AMPD2 gene. Previous sequence analysis identified both members of the GSTM family and the GNAI3 gene within a cloned 120-kb region surrounding the AMPD2 locus. We show here that the GNAT2 gene, which is inactive in the fibroblastic cells, lies within the 20 kb separating the transcriptionally active GNAI3 and AMPD2 genes. We map most gene ends by sequence comparison with human homologs; one is inferred from the presence of an unmethylated CpG island. This Chinese hamster locus corresponds to a region of conserved linkage between human Chr 1 (locus 1p13) and mouse Chr 3 (position 52.5 cM), where Gnai-3 and Gnat-2 have been mapped. The AMPD2 gene is presently unlocalized in human genome; its proposed position on mouse Chr 3 is at 53.4 cM. Our results, obtained by physical mapping, strongly suggest that the order and possibly the tight linkage of these genes are conserved on all three genomes.

The highly conserved Chinese hamster GNAI3 gene maps less than 60 kb from the AMPD2 gene and lacks the intronic U6 snRNA present in its human counterpart.

The identity of a gene coamplified with the adenylate deaminase 2 gene (AMPD2) in coformycin-resistant cells was determined by analysis of its genomic sequence. Sequence comparisons reveal a significant homology with the 3' terminal part of the gene encoding the alpha i3 subunit of Gi proteins from several species (GNAI3). Identification of the gene was confirmed by Western blot analysis of its products. A precise sequence comparison was performed with the human genomic sequence. It showed that conservation remains important in noncoding exons as well as in introns. However, sequences corresponding to combined U6 snRNA and E protein pseudogene, previously identified inside intron 7 of the human gene, were not found in the Chinese hamster gene. GNAI3 is mapped to a region of conserved linkage between human chromosome 1 (locus 1p13) and mouse chromosome 3 (at 48.4 cM). The Chinese hamster GNAI3 gene maps to chromosome 1 within a 120-kb fragment that also comprises the AMPD2 and GSTM genes.

[Gene amplification and chromosomal rearrangements during acquisition of cellular resistance to the antimetabolite coformycin]. / Amplification génique et réarrangements chromosomiques lors de l'acquisition de la résistance cellulaire à un antimétabolite, la coformycine.

We studied the early stages of gene amplification in a Chinese hamster cell line and we show that two distinct mechanisms can operate at a single locus. Both of them rely on an unequal segregation of gene copies at mitosis. We conclude that cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere that lead to further breaks in mitosis, have a key role in the coupling of gene amplification and genome remodeling. Rearrangements are first limited to a single chromosome but can then potentially spread to any additional chromosome. Occasionally, a sequence containing the selected gene can be looped out, generating a "double minute" and thus initiating an independent process of extrachromosomal amplification.

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells.

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.

Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Two-colour in situ hybridization with probes for two co-amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase-long inverted repeats. Their organization is remarkably well accounted for if breakage-fusion-bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.