RESUMO
Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.
Assuntos
Hidrolases Anidrido Ácido/genética , Sítios Frágeis do Cromossomo/genética , Fragilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Neoplasias/genética , Origem de Replicação/genética , Linhagem Celular , Quebra Cromossômica , Fragilidade Cromossômica/genética , Replicação do DNA/genética , Fibroblastos , Genes Supressores de Tumor , Loci Gênicos/genética , Humanos , Linfócitos/metabolismo , Modelos Biológicos , Especificidade de ÓrgãosRESUMO
Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.
Assuntos
Cromatina/genética , Sítios Frágeis do Cromossomo/genética , Instabilidade Genômica/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Replicação do DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Mitose/genética , Proteínas Quinases/genética , Origem de Replicação/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Common fragile sites (CFSs) are large chromosomal regions prone to breakage upon replication stress that are considered a driving force of oncogenesis. CFSs were long believed to contain sequences blocking fork progression, thus impeding replication completion and leading to DNA breaks upon chromosome condensation. However, recent studies show that delayed completion of DNA replication instead depends on a regional paucity in initiation events. Because the distribution and the timing of these events are cell type dependent, different chromosomal regions can be committed to fragility in different cell types. These new data reveal the epigenetic nature of CFSs and open the way to a reevaluation of the role played by these sites in the formation of chromosome rearrangements found in tumors from different tissues.
Assuntos
Sítios Frágeis do Cromossomo , Instabilidade Genômica , Animais , DNA/genética , DNA/metabolismo , Replicação do DNA , Epigênese Genética , Humanos , Transcrição GênicaRESUMO
Common fragile sites (CFSs) are large chromosomal regions long identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs came from their key role in the formation of DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was notably correlated with the appearance of genome instability in precancerous lesions and during tumor progression. Identification of the molecular mechanisms responsible for their instability therefore represents a major challenge. A number of data show that breaks result from mitotic entry before replication completion but the mechanisms responsible for such delayed replication of CFSs and relaxed checkpoint surveillance are still debated. In addition, clues to the molecular events leading to breakage just start to emerge. We present here the results of recent reports addressing these questions.
Assuntos
Sítios Frágeis do Cromossomo , Instabilidade Genômica , Neoplasias/genética , Animais , Ciclo Celular , Dano ao DNA , Replicação do DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologiaRESUMO
The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.
Assuntos
Duplicação Cromossômica , Amplificação de Genes , Neoplasias/genética , Linhagem Celular Tumoral , Sítios Frágeis do Cromossomo , Cromossomos Humanos Par 17 , Genes myc , Humanos , Hibridização in Situ Fluorescente , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.
Assuntos
Cromatina/metabolismo , Replicação do DNA/fisiologia , Movimento , Origem de Replicação/genética , Animais , Linhagem Celular , Cromatina/genética , Cricetinae , Cricetulus , DNA/biossíntese , DNA/genética , Fase G1 , Matriz Nuclear/metabolismo , Fase S , Fatores de TempoRESUMO
Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Humanos , Fase S , Sítios Frágeis do Cromossomo/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNARESUMO
In eukaryotes, only a fraction of replication origins fire at each S phase. Local histone acetylation was proposed to control firing efficiency of origins, but conflicting results were obtained. We report that local histone acetylation does not reflect origin efficiencies along the adenosine monophosphate deaminase 2 locus in mammalian fibroblasts. Reciprocally, modulation of origin efficiency does not affect acetylation. However, treatment with a deacetylase inhibitor changes the initiation pattern. We demonstrate that this treatment alters pyrimidine biosynthesis and decreases fork speed, which recruits latent origins. Our findings reconcile results that seemed inconsistent and reveal an unsuspected effect of deacetylase inhibitors on replication dynamics.
Assuntos
Replicação do DNA , Histonas/metabolismo , Nucleotídeos/metabolismo , Origem de Replicação , Acetilação , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Transcrição GênicaRESUMO
Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.
Assuntos
Sítios Frágeis do Cromossomo/genética , Período de Replicação do DNA/genética , Instabilidade Genômica , Fase S/genética , Terminação da Transcrição Genética , Linhagem Celular , Humanos , Origem de Replicação , Transcrição GênicaRESUMO
The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3' end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5' exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells.
Assuntos
Fusão Gênica Artificial , DNA Ribossômico/genética , Genes myc , Genes de RNAr , Poliadenilação , RNA Polimerase I/metabolismo , RNA Mensageiro/metabolismo , Trans-Splicing , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , RNA Mensageiro/químicaRESUMO
We are investigating the mechanism responsible for the overexpression of the keratin 18 (K18) gene in tumorigenic clones from the SW613-S human colon carcinoma cell line, as compared with non-tumorigenic clones. We have previously shown that this mechanism affects the minimal K18 promoter (TATA box and initiation site). We report here that treatment of the cells with histone deacetylase inhibitors stimulates the activity of the promoter in non-tumorigenic cells but has no effect in tumorigenic cells, resulting in a comparable activity of the promoter in both cell types. The adenovirus E1A protein inhibits the activity of the K18 promoter specifically in tumorigenic cells. This inhibition can be reversed by an excess of CBP protein. The conserved region 1 (CR1) of E1A, which is involved in the interaction with the CBP/p300 co-activators, is necessary to the inhibitory capacity of E1A. A 79 amino acid long N-terminal fragment of E1A, encompassing the two domains of E1A necessary and sufficient for binding to CBP (N-terminus and CR1), has the same differential inhibitory capacity on the K18 promoter as wild-type E1A. Forced recruitment of GAL4-CBP fusion proteins to the K18 promoter results in a greater stimulation of its activity in non-tumorigenic than in tumorigenic cells. The histone acetyltransferase activity of CBP is essential for this differential stimulation and the presence of the CBP2 domain greatly augments the activation capacity of the fusion protein. Chromatin immunoprecipitation experiments carried out with anti-acetylated histone antibodies showed no difference in the level of histone acetylation in the region of the K18 promoter between the two cell types. The structure of chromatin in the promoter region is similar in tumorigenic and non-tumorigenic cells, as determined by mapping of DNase I hypersensitive sites and probing the accessibility of the DNA to restriction endonucleases. From all these results we conclude that alteration of an acetylation mechanism involving the CBP (or p300) protein and acting on a non-histone substrate is responsible for the higher activity of the K18 promoter in tumorigenic cells of the SW613-S cell line.
Assuntos
Carcinoma/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Queratinas/genética , Ativação Transcricional , Acetilação , Acetiltransferases/fisiologia , Proteínas E1A de Adenovirus/farmacologia , Carcinoma/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/fisiologia , Cromatina/ultraestrutura , Células Clonais , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Acetiltransferases , Inibidores de Histona Desacetilases , Histonas/metabolismo , Humanos , Queratinas/biossíntese , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Transativadores/metabolismo , Fatores de Transcrição , Células Tumorais Cultivadas , Fatores de Transcrição de p300-CBPRESUMO
Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates.
Assuntos
Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Proteínas Quinases/deficiência , Origem de Replicação , Transdução de Sinais , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Quinase 1 do Ponto de Checagem , Reparo do DNA , Desoxirribonucleosídeos/metabolismo , Humanos , Proteína Homóloga a MRE11 , Proteínas Quinases/metabolismoRESUMO
BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12-25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes BRCA1/fisiologia , Queratinas/genética , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Oxirredutases do Álcool , Western Blotting , Linhagem Celular Tumoral , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Queratina-18 , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/fisiologia , Deleção de SequênciaRESUMO
Neuroblastomas, tumors of the sympathetic nervous system, account for 7-10% of the cancers of childhood. Genetic studies have shown, and this study has confirmed, that neuroblastomas are very heterogeneous; no single genetic change common to all neuroblastomas has yet been identified. One genetic aberration found frequently in this pediatric tumor is MYCN gene amplification. Recently we identified a new subset of tumors showing MYCN gain (small increases in gene number arising from unbalanced translocation). To investigate whether gain precedes amplification or is an independent event, we surveyed 200 primary tumors for MYCN copy number with fluorescence in situ hybridization; 152 of 200 (76%) were MYCN single-copy tumors, whereas 48 of 200 (24%) tumors harbored MYCN abnormalities: 36 of the 48 (75%) had MYCN amplification and 12 (25%) had MYCN gain. Among the 36 with MYCN amplified gene, we found four that also showed gain. In three tumors exhibiting simultaneous gain and amplification, these two events were detected in neighboring cells. In the fourth case we detected only MYCN gain in metastatic neuroblasts in the bone marrow, but both MYCN amplification and gain in the primary tumor. The detailed study of these four cases suggests that there may be several different mechanisms leading to increase in MYCN copy number. Further studies in other human malignancies are necessary to determine whether simultaneous gain and amplification are specific to neuroblastoma or constitute a general mechanism by which tumor cells can acquire selective growth advantage.
Assuntos
Genes myc , Neuroblastoma/genética , Criança , Células Clonais/patologia , Amplificação de Genes , Heterogeneidade Genética , Humanos , Hibridização in Situ Fluorescente , Modelos Genéticos , Neuroblastoma/patologiaRESUMO
We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myc-dependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2G1mycP2Tu1 cell lines, respectively), activate the apoptotic machinery in response to the treatment with etoposide, doxorubicin and vitamin D3, which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12A1 and B3 cell lines.
Assuntos
Apoptose/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Genes myc , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Linhagem Celular Tumoral , Colecalciferol/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Doxorrubicina/farmacologia , Endodesoxirribonucleases/metabolismo , Ativação Enzimática , Etoposídeo/farmacologia , Expressão Gênica , Humanos , Transfecção , Receptor fas/metabolismoRESUMO
Cytogenetics has clearly established the key role chromosomal rearrangements play in neoplastic initiation and progression. Investigation methods have evolved considerably from banding analysis of chromosome morphology to fluorescence in situ hybridization (FISH) and now to comparative genomic hybridization (CGH) on chromosomes or micro-array analysis of DNA. In addition to its contributions to the description, prognosis and understanding of oncogenesis and tumor progression, cytogenomics provides the information required for the rational use of new targeted therapies among which Imatimib is the most achieved example.
Assuntos
Aberrações Cromossômicas , Genômica/tendências , Neoplasias/genética , Análise Citogenética , Bases de Dados Genéticas , Genoma Humano , Genômica/métodos , Humanos , Hibridização in Situ Fluorescente , Neoplasias/diagnóstico , Neoplasias/terapia , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.
Assuntos
Sítios Frágeis do Cromossomo , Células Epiteliais/ultraestrutura , Células Eritroides/ultraestrutura , Neoplasias/genética , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Eritroides/metabolismo , Células HCT116 , Humanos , Células K562 , Transcrição GênicaRESUMO
The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.
Assuntos
Técnicas Citológicas/métodos , Replicação do DNA , Animais , Linhagem Celular , Humanos , Mamíferos , Coloração e Rotulagem/métodosRESUMO
Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.
Assuntos
Síndrome de Bloom/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Pirimidinas/metabolismo , RecQ Helicases/deficiência , Western Blotting , Linhagem Celular , Citidina Desaminase/metabolismo , Primers do DNA/genética , Humanos , Análise em Microsséries , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Troca de Cromátide Irmã/genética , Estatísticas não ParamétricasRESUMO
Common fragile sites have been mapped primarily in lymphocytes, but recent analyses show that the setting of these sites relies on cell type-dependent replication programs. Using a new approach, we molecularly mapped common fragile sites in human fibroblasts and showed that commitment to fragility depends on similar replication features in fibroblasts and lymphocytes, although different loci are committed in each cell type. Notably, the common fragile sites that we identified overlapped heretofore unexplained deletion clusters observed in tumors.