RESUMEN
Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN/genética , Proteínas Nucleares/metabolismo , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular , Cromatina/metabolismo , Daño del ADN/genética , ADN Helicasas/metabolismo , Proteínas Nucleares/genética , Mutación Puntual , Unión Proteica , Recombinasa Rad51/metabolismoRESUMEN
R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.
Asunto(s)
Reparación del ADN , Inestabilidad Genómica , Daño del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Genoma Humano , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.
Asunto(s)
Reparación del ADN , Células Endoteliales/metabolismo , Homeostasis , PPAR gamma/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Inestabilidad Genómica , Células HEK293 , Humanos , Modelos Biológicos , Unión Proteica , Arteria Pulmonar/patología , Transducción de Señal , UbiquitinaciónRESUMEN
RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Muerte Celular , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Letales/genética , Péptidos y Proteínas de Señalización Intracelular , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Ribonucleótido Reductasas/antagonistas & inhibidores , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada/genética , N-Metiltransferasa de Histona-Lisina , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/metabolismo , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.
Asunto(s)
Daño del ADN , Estrógenos/toxicidad , Mutágenos/metabolismo , Transcripción Genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Humanos , Células MCF-7 , ARN Mensajero/metabolismoRESUMEN
R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded (ss) DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin (Ig) class switching, and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. We discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability, and their potential contribution to the origin of diseases including cancer and neurodegenerative disorders.
Asunto(s)
ADN de Cadena Simple/fisiología , Inestabilidad Genómica , Animales , Cromatina/fisiología , Cromatina/ultraestructura , Daño del ADN , Reparación del ADN , Epigénesis Genética , HumanosRESUMEN
Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Reparación del ADN , Proteínas Nucleares/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Daño del ADN , Replicación del ADN/genética , Mutación/genética , Proteínas Nucleares/genética , Unión Proteica , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
The Ubc9 SUMO-conjugating enzyme and the Siz1 SUMO ligase sumoylate several repair and recombination proteins, including PCNA. Sumoylated PCNA binds Srs2, a helicase counteracting certain recombination events. Here we show that ubc9 mutants depend on checkpoint, recombination, and replication genes for growth. ubc9 cells maintain stalled-fork stability but exhibit a Rad51-dependent accumulation of cruciform structures during replication of damaged templates. Mutations in the Mms21 SUMO ligase resemble the ubc9 mutations. However, siz1, srs2, or pcna mutants altered in sumoylation do not exhibit the ubc9/mms21 phenotype. Like ubc9/mms21 mutants, sgs1 and top3 mutants also accumulate X molecules at damaged forks, and Sgs1/BLM is sumoylated. We propose that Ubc9 and Mms21 act in concert with Sgs1 to resolve the X structures formed during replication. Our results indicate that Ubc9- and Mms21-mediated sumoylation functions as a regulatory mechanism, different from that of replication checkpoints, to prevent pathological accumulation of cruciform structures at damaged forks.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN de Hongos/fisiología , Recombinación Genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Epigénesis Genética , Genes Fúngicos , Mutación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , RecQ Helicasas , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The entry into meiosis is characterized by a lengthy premeiotic S phase and a reorganization of the nuclear architecture. Analysis of centromere and telomere dynamics in wild-type Saccharomyces cerevisiae meiosis suggests that resolution of vegetative centromere and telomere clusters are independent events differently connected to premeiotic S phase. Absence of the B-type cyclin Clb5 or the Set1 histone methyltransferase leads to a delay of premeiotic S phase by separate mechanisms. In clb5Delta cells, centromere cluster resolution appears normal, whereas dissolution of the vegetative telomere clusters is impaired and meiosis-specific clustering of telomeres, i.e. bouquet formation, is grossly delayed. In set1Delta cells, centromere and telomere redistribution are both impaired and bouquet nuclei are absent, despite proper location of the meiosis-specific telomere protein Ndj1. Thus, centromere and telomere redistribution at the onset of prophase I is differentially regulated, with centromere dispersion occurring independently of premeiotic S phase. The normal kinetics of dissolution of the vegetative telomere clusters in a set1Delta mec1-1 mutant suggests the presence of a checkpoint that limits the dispersion of telomeres in absence of Set1.
Asunto(s)
Centrómero/metabolismo , Ciclina B/genética , Proteínas de Unión al ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Telómero/metabolismo , Factores de Transcripción/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Emparejamiento Cromosómico/genética , Ciclina B/deficiencia , Proteínas de Unión al ADN/deficiencia , Epistasis Genética , Silenciador del Gen , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Péptidos y Proteínas de Señalización Intracelular , Meiosis/genética , Proteína Metiltransferasas , Proteínas Serina-Treonina Quinasas , Fase S/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismoRESUMEN
The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3. Inactivation of the SET1 gene in a diploid leads to a sporulation defect. We have studied various processes that take place during meiotic differentiation in set1delta diploid cells. The absence of Set1 leads to a delay of meiotic S-phase onset, which reflects a defect in DNA replication initiation. The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain. In addition, set1delta displays a severe impairment of the DNA double-strand break formation, which is not only the consequence of the replication delay. Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1. In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3-K4. Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase-dependent and -independent Set1 functions.