RESUMEN
The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Ratones , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Recombinasas/metabolismo , Sumoilación , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , Quinasa Tipo Polo 1RESUMEN
Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.
Asunto(s)
Replicación del ADN , Ubiquitinación , Humanos , Reparación del ADN , ADN/metabolismoRESUMEN
Copy number variants (CNVs) are an important component of genomic variation in humans and other mammals. Similar de novo deletions and duplications, or copy number changes (CNCs), are now known to be a major cause of genetic and developmental disorders and to arise somatically in many cancers. A major mechanism leading to both CNVs and disease-associated CNCs is meiotic unequal crossing over, or nonallelic homologous recombination (NAHR), mediated by flanking repeated sequences or segmental duplications. Others appear to involve nonhomologous end joining (NHEJ) or aberrant replication suggesting a mitotic cell origin. Here we show that aphidicolin-induced replication stress in normal human cells leads to a high frequency of CNCs of tens to thousands of kilobases across the human genome that closely resemble CNVs and disease-associated CNCs. Most deletion and duplication breakpoint junctions were characterized by short (<6 bp) microhomologies, consistent with the hypothesis that these rearrangements were formed by NHEJ or a replication-coupled process, such as template switching. This is a previously unrecognized consequence of replication stress and suggests that replication fork stalling and subsequent error-prone repair are important mechanisms in the formation of CNVs and pathogenic CNCs in humans.
Asunto(s)
Replicación del ADN/genética , Dosificación de Gen , Variación Genética , Genoma Humano , Afidicolina/farmacología , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Estudio de Asociación del Genoma Completo , Humanos , Recombinación GenéticaRESUMEN
Common fragile sites (CFSs) are loci that preferentially exhibit metaphase chromosome gaps and breaks after partial inhibition of DNA synthesis. The fragile site FRA3B, which lies within the FHIT tumor-suppressor gene, is a site of frequent heterozygous and homozygous deletions in many cancer cells and precancerous lesions. The great majority of FHIT and other CFS-associated gene rearrangements in tumors are submicroscopic, intralocus deletions of hundreds of kilobases that often result in inactivation of associated genes. Although CFS instability leads to chromosome gaps and breaks and translocations, there has been no direct evidence showing that CFS instability or replication stress can generate large submicroscopic deletions of the type seen in cancer cells. Here, we have produced FHIT/FRA3B deletions closely resembling those in tumors by exposing human-mouse chromosome 3 somatic hybrid cells to aphidicolin-mediated replication stress. Clonal cell populations were analyzed for deletions by using PCR, array comparative genomic hybridization (aCGH), and FISH. Thirteen percent to 23% of clones exhibited submicroscopic FHIT deletions spanning approximately 200-600 kb within FRA3B. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus, with a 2- to 12-fold reduction in metaphase gaps and breaks compared with controls. Sequence analysis showed no regions of homology at breakpoints and suggests involvement of NHEJ in generating the deletions. Our results demonstrate that replication stress induces a remarkably high frequency of tumor-like microdeletions that reduce fragility at a CFS in cultured cells and suggests that similar conditions during tumor formation lead to intralocus deletion and inactivation of genes at CFSs and perhaps elsewhere in the genome.
Asunto(s)
Ácido Anhídrido Hidrolasas/biosíntesis , Ácido Anhídrido Hidrolasas/genética , Replicación del ADN , Eliminación de Gen , Regulación de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias/genética , Animales , Cromosomas/ultraestructura , Genoma Humano , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Ratones , Modelos Genéticos , Neoplasias/metabolismo , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
Studies of tickborne illness have benefited from interactions between scientists and community members. Most participants in community science projects are well-educated adults, but there are anticipated benefits from engaging younger students in research. We evaluated whether an outreach experience for rural middle-school students promoted student interest in science and resulted in the generation of samples that could be used for tick testing to assess disease risk. Middle-school students from 78 Wisconsin communities developed interdisciplinary hypotheses about the spread of Lyme disease, identified ticks, and extracted DNA from ticks to assess the prevalence of pathogens Borrelia burgdorferi, Anaplasma phagocytophillium, and Babesia microti. As a result of this intervention, students were able to successfully complete the research protocol and explain the rationale for completing the experiment. Of student participants, 84.7% reported no difficulty completing the protocol, 66% of the student samples gave reliable PCR results, and 76% of students reported interest in participating in similar experiments. Our study shows that tick outreach programs that incorporate community-based science promote knowledge about Lyme disease, facilitate engagement between students and scientists, and generate samples that can be successfully utilized for pathogen testing.
RESUMEN
Cytokine production is a critical component of cell-extrinsic responses to DNA damage and cellular senescence. Here, we demonstrated that expression of the gene encoding interleukin-19 (IL-19) was enhanced by DNA damage through pathways mediated by c-Jun amino-terminal kinase (JNK) and cGAS-STING and that IL19 expression was required for the subsequent production of the cytokines IL-1, IL-6, and IL-8. IL19 expression was stimulated by diverse cellular stresses, including inhibition of the DNA replication checkpoint kinase ATR (ataxia telangiectasia and Rad3-related protein), oncogene expression, replicative exhaustion, oxidative stress, and DNA double-strand breaks. Unlike the production of IL-6 and IL-8, IL19 expression was not affected by abrogation of signaling by the IL-1 receptor (IL-1R) or the mitogen-activated protein kinase p38. Instead, the DNA damageinduced production of IL-1, IL-6, and IL-8 was substantially reduced by suppression of IL19 expression. The signaling pathways required to stimulate IL19 expression selectively depended on the type of DNA-damaging agent. Reactive oxygen species and the ASK1-JNK pathway were critical for responses to ionizing radiation (IR), whereas the cGAS-STING pathway stimulated IL19 expression in response to either IR or ATR inhibition. Whereas induction of IL1, IL6, and IL8 by IR depended on IL19 expression, the cGAS-STINGdependent induction of the immune checkpoint gene PDL1 after IR and ATR inhibition was independent of IL19. Together, these results suggest that IL-19 production by diverse pathways forms a distinct cytokine regulatory arm of the response to DNA damage.
Asunto(s)
Daño del ADN , Interleucinas/metabolismo , Proteínas de la Membrana , Transducción de Señal , Animales , Citocinas/genética , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/genética , Ratones , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
The ATR checkpoint pathway responds to DNA damage during the S/G2 phases of the cell cycle and is activated early in tumorigenesis. Investigation of ATR's role in development and tumorigenesis is complicated by the lethality of homozygous knockout mice and the limited effects of heterozygous deficiency. To overcome this limitation, we sought to create mice with a hypomorphic Atr mutation based on the ATR mutation in the human disease Seckel syndrome-1 (SCKL1). Homozygous SCKL1 mice were generated by targeted knock-in of the A --> G SCKL1 mutation. Western blot and RT-PCR analysis established that homozygotes have no reduction in Atr protein or increase in missplicing as is seen in humans. Thus, the A --> G substitution alone is not sufficient to reproduce in mice the effects that are seen in humans. However, homozygous SCKL1 mice that retain the neo cassette used for targeting have an estimated 66-82% reduction in total Atr protein levels due to missplicing into the neo cassette. Under conditions of APH-induced replication stress, primary fibroblasts from homozygous mice displayed an increase in overall chromosome damage and an increase in gaps and breaks at specific common fragile sites. In addition, mutant cells display a significant delay in checkpoint induction and an increase in DNA damage as assayed by Chk1 phosphorylation and gamma-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular , Daño del ADN , Enanismo/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Enanismo/metabolismo , Enanismo/fisiopatología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Mutación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Common fragile sites (CFSs) are loci that are especially prone to forming gaps and breaks on metaphase chromosomes under conditions of replication stress. Although much has been learned about the cellular responses to gaps and breaks at CFSs, less is known about what makes these sites inherently unstable. CFS sequences are highly conserved in mammalian evolution and contain a number of sequence motifs that are hypothesized to contribute to their instability. To examine the role of CFS sequences in chromosome breakage, we stably transfected two BACs containing FRA3B sequences and two nonCFS control BACs containing similar sequence content into HCT116 cells and isolated cell clones with BACs integrated at ectopic sites. Integrated BACs were present at just a few to several hundred contiguous copies. Cell clones containing integrated FRA3B BACs showed a significant, three to sevenfold increase in aphidicolin-induced gaps and breaks at the integration site as compared to control BACs. Furthermore, many FRA3B integration sites displayed additional chromosome rearrangements associated with CFS instability. Clones were examined for replication timing and it was found that the integrated FRA3B sequences were not dependent on late replication for their fragility. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic nonfragile loci is sufficient to recapitulate the instability found at CFSs. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs.
Asunto(s)
Ácido Anhídrido Hidrolasas/fisiología , Sitios Frágiles del Cromosoma , Fragilidad Cromosómica/genética , Proteínas de Neoplasias/fisiología , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Cromosomas Humanos/genética , Replicación del ADN , Biblioteca Genómica , Células HCT116 , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Common fragile sites are large chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed. Fragile site instability in cultured cells is well documented and includes gaps and breaks on metaphase chromosomes, translocation and deletions breakpoints, and sister chromosome exchanges. In recent years, much has been learned about the genomic structure at fragile sites and the cellular mechanisms that monitor their stability. The study of fragile sites has merged with that of cell cycle checkpoints and DNA repair, with multiple proteins from these pathways implicated in fragile site stability, including ATR, BRCA1, CHK1, and RAD51. Since their discovery, fragile sites have been implicated in constitutional and cancer chromosome rearrangements in vivo and recent studies suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early tumorigenesis. Here we review the relationship of fragile sites to chromosome rearrangements, particularly in tumor cells, and discuss the mechanisms that may be involved.
Asunto(s)
Sitios Frágiles del Cromosoma , Neoplasias/genética , Translocación Genética , Fragilidad Cromosómica , Cromosomas Humanos , Inestabilidad Genómica , Humanos , Modelos Genéticos , Células Tumorales CultivadasRESUMEN
Purpose: PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression.Experimental Design: Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell cycle, genome instability, and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived xenograft (PDX) model.Results: PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum tolerated dose (MTD) in a BRCA2MUT PDX. PARPi treatment increased pATR and pCHK1, indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared with single-agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA2MUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission.Conclusions: PARPi causes increased reliance on ATR/CHK1 for genome stability, and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models. Clin Cancer Res; 23(12); 3097-108. ©2016 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteína BRCA1/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Inestabilidad Genómica/efectos de los fármacos , Humanos , Indoles , Ratones , Terapia Molecular Dirigida , Morfolinas , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Sulfonamidas , Sulfóxidos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Mutación , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Oncogenes , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Daño del ADN , Fibrosarcoma/genética , Fibrosarcoma/terapia , Genes p53 , Genes ras , Homeostasis , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Neoplasias Experimentales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés FisiológicoRESUMEN
Previous studies indicate that oncogenic stress activates the ATR-Chk1 pathway. Here, we show that ATR-Chk1 pathway engagement is essential for limiting genomic instability following oncogenic Ras transformation. ATR pathway inhibition in combination with oncogenic Ras expression synergistically increased genomic instability, as quantified by chromatid breaks, sister chromatid exchanges, and H2AX phosphorylation. This level of instability was significantly greater than that observed following ATR suppression in untransformed control cells. In addition, consistent with a deficiency in long-term genome maintenance, hypomorphic ATR pathway reduction to 16% of normal levels was synthetic lethal with oncogenic Ras expression in cultured cells. Notably, elevated genomic instability and synthetic lethality following suppression of ATR were not due to accelerated cycling rates in Ras-transformed cells, indicating that these synergistic effects were generated on a per-cell-cycle basis. In contrast to the synthetic lethal effects of hypomorphic ATR suppression, subtle reduction of ATR expression (haploinsufficiency) in combination with endogenous levels of K-ras(G12D) expression elevated the incidence of lung adenocarcinoma, spindle cell sarcoma, and thymic lymphoma in p53 heterozygous mice. K-ras(G12D)-induced tumorigenesis in ATR(+/-)p53(+/-) mice was associated with intrachromosomal deletions and loss of wild-type p53. These findings indicate that synergistic increases in genomic instability following ATR reduction in oncogenic Ras-transformed cells can produce 2 distinct biological outcomes: synthetic lethality upon significant suppression of ATR expression and tumor promotion in the context of ATR haploinsufficiency. These results highlight the importance of the ATR pathway both as a barrier to malignant progression and as a potential target for cancer treatment.
Asunto(s)
Proteínas de Ciclo Celular/genética , Genes ras/genética , Inestabilidad Genómica , Neoplasias Experimentales/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Antineoplásicos Hormonales/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/genética , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Recombinación Genética/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tamoxifeno/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Poleta), REV1, and zeta (Polzeta) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polzeta are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polzeta facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Poleta, whereas RAD18 plus Poleta, REV1, and Polzeta are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.