RESUMEN
One of a major function of programmed cell death (apoptosis) is the removal of cells which suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes which, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS-promoters at both ends which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS-promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, seven genes were identified which suppressed the p53-induced apoptosis. In four mutants, the suppression effect resulted from single genes activated by one UAS-promoter (Pka-R2, Rga, crol, Spt5). In the other three (Orct2, Polr2M, stg), deleting either UAS-promoter eliminated the suppression effect. In qPCR experiments we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eucaryotic genomes there are co-expressed gene clusters. Three of the DEP insertion mutants are included and two are in close vicinity of separate co-expressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.
RESUMEN
We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN/genética , Subunidades de Proteína/genética , Proteína de Replicación A/genética , Ubiquitina-Proteína Ligasas/genética , Cromatina/química , Cromatina/metabolismo , ADN/química , Daño del ADN , Células HeLa , Recombinación Homóloga , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Subunidades de Proteína/metabolismo , Proteína de Replicación A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/metabolismo , Recombinación Homóloga , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Secuencias de Aminoácidos , Animales , Carcinoma Epitelial de Ovario , Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades/genética , Replicación del ADN , ADN Polimerasa Dirigida por ADN/deficiencia , Pérdida del Embrión , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Femenino , Inestabilidad Genómica , Recombinación Homóloga/genética , Humanos , Ratones , Terapia Molecular Dirigida , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Unión Proteica , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación/genética , ADN Polimerasa thetaRESUMEN
Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Proteínas de Arabidopsis , Línea Celular , Replicación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Endopeptidasas/metabolismo , Humanos , Estructura Terciaria de Proteína , Intercambio de Cromátides Hermanas , Ubiquitina-Proteína Ligasas/fisiología , Proteasas Ubiquitina-EspecíficasRESUMEN
Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN/fisiología , Inestabilidad Genómica/fisiología , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Daño del ADN/fisiología , ADN Helicasas/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Datos de Secuencia Molecular , Osteosarcoma , Unión Proteica/fisiología , Recombinación Genética/fisiología , Intercambio de Cromátides Hermanas/fisiología , Ubiquitinación/fisiologíaRESUMEN
DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.
Asunto(s)
Roturas del ADN de Doble Cadena , Antígeno Nuclear de Célula en Proliferación/metabolismo , Sumoilación , Replicación del ADN , Histonas/metabolismo , Recombinación Homóloga , Humanos , Mutación , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Proteína SUMO-1/metabolismoRESUMEN
Wolf-Hirschhorn syndrome (WHS) is a malformation syndrome associated with growth retardation, mental retardation, and immunodeficiency resulting from a hemizygous deletion of the short arm of chromosome 4, called the WHS critical region (WHSC). The WHSC1 gene is located in this region, and its loss is believed to be responsible for a number of WHS characteristics. We identified WHSC1 in a genetic screen for genes involved in responding to replication stress, linking Wolf-Hirschhorn syndrome to the DNA damage response (DDR). Here, we report that the WHSC1 protein is a member of the DDR pathway. WHSC1 localizes to sites of DNA damage and replication stress and is required for resistance to many DNA-damaging and replication stress-inducing agents. Through its SET domain, WHSC1 regulates the methylation status of the histone H4 K20 residue and is required for the recruitment of 53BP1 to sites of DNA damage. We propose that Wolf-Hirschhorn syndrome results from a defect in the DDR.
Asunto(s)
Daño del ADN , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Represoras/metabolismo , Síndrome de Wolf-Hirschhorn/metabolismo , Síndrome de Wolf-Hirschhorn/patología , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Replicación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/deficiencia , Histonas/metabolismo , Humanos , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/metabolismo , Metilación/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteína de Replicación A/metabolismo , Proteínas Represoras/deficiencia , Estrés Fisiológico/efectos de los fármacos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
In the yeast Saccharomyces cerevisiae, the Rad6-Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6-Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.
Asunto(s)
Daño del ADN , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , ADN Helicasas/química , Replicación del ADN , Proteínas de Unión al ADN/química , Humanos , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.
Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
Human Ape2 protein has 3' phosphodiesterase activity for processing 3'-damaged DNA termini, 3'-5' exonuclease activity that supports removal of mismatched nucleotides from the 3'-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3'-5' exonuclease and 3' phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3'-damaged termini but also by removing 3'-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Exodesoxirribonucleasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Adenina/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Endonucleasas , Humanos , Peróxido de Hidrógeno/farmacología , Enzimas Multifuncionales , Oxidación-Reducción , Antígeno Nuclear de Célula en Proliferación/análisisRESUMEN
Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Adenosina Trifosfatasas/metabolismo , Línea Celular , ADN Helicasas , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/efectos de la radiación , Prueba de Complementación Genética , Humanos , Ligasas/metabolismo , Lisina/metabolismo , Metilmetanosulfonato/farmacología , Mutación/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/efectos de la radiación , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/efectos de la radiación , Rayos UltravioletaRESUMEN
Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.
Asunto(s)
ADN Helicasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Línea Celular , ADN/genética , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/aislamiento & purificaciónRESUMEN
When injected into the cerebral ventricles, the somatostatin analog, octreotide (OCT) elicits prompt drinking, vasopressin secretion and increases in blood pressure that are attributed to the activation of the intracerebral angiotensinergic system. In addition, OCT induces sleep responses that might be mediated by an inhibition of hypothalamic neurons producing growth hormone-releasing hormone (GHRH). OCT (0.02 microg in 0.2 microl) was microinjected into various brain sites to determine the structures inducing drinking and/or sleep suppression in response to OCT in rats. Drinking (>1 ml water in 10 min) was elicited in 17 rats out of 86 tested. The positive drinking sites resided in or around the subfornical organ (SFO) and the paraventricular nucleus. Both structures are part of the reported angiotensinergic dipsogenic circuit of the brain. These microinjections failed to elicit consistent sleep effects. Sleep suppression (>10% recording time in hour 1) was observed after injection of OCT either into the arcuate nucleus (n=7), where the majority of GHRHergic neurons reside, or into the medial preoptic area/anterior hypothalamus (n=8), where GHRH acts to promote sleep. Administration of OCT into far lateral sites of the lateral preoptic area and lateral hypothalamus stimulated sleep in hour 1 (n=10), perhaps via inhibiting cholinergic neurons previously implicated in arousal. The results are consistent with the hypothesis that somatostatin is involved in the regulation of both water intake and sleep, and suggest that different structures, and therefore different somatostatinergic neuronal pools, mediate these actions.