RESUMEN
PARPs play fundamental roles in multiple DNA damage recognition and repair pathways. Persistent nuclear PARP activation causes cellular NAD+ depletion and exacerbates cellular aging. However, very little is known about mitochondrial PARP (mtPARP) and poly ADP-ribosylation (PARylation). The existence of mtPARP is controversial, and the biological roles of mtPARP-induced mitochondrial PARylation are unclear. Here, we demonstrate the presence of PARP1 and PARylation in purified mitochondria. The addition of the PARP1 substrate NAD+ to isolated mitochondria induced PARylation, which was suppressed by treatment with the inhibitor olaparib. Mitochondrial PARylation was also evaluated by enzymatic labeling of terminal ADP-ribose (ELTA). To further confirm the presence of mtPARP1, we evaluated mitochondrial nucleoid PARylation by ADP ribose-chromatin affinity purification (ADPr-ChAP) and PARP1 chromatin immunoprecipitation (ChIP). We observed that NAD+ stimulated PARylation and TFAM occupancy on the mtDNA regulatory region D-loop, inducing mtDNA transcription. These findings suggest that PARP1 is integrally involved in mitochondrial PARylation and that NAD+-dependent mtPARP1 activity contributes to mtDNA transcriptional regulation.
Asunto(s)
NAD , Poli ADP Ribosilación , NAD/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Mitocondrias/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismoRESUMEN
Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser-induced DNA double-strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non-homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN's affinity for RPA, likely increasing its long-range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2-dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Recombinación Homóloga , Transducción de Señal/genética , Helicasa del Síndrome de Werner/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/genética , ADN/metabolismo , Células HEK293 , Humanos , Fosforilación/genética , Proteína de Replicación A/metabolismo , Serina/metabolismo , Transfección , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicasa del Síndrome de Werner/genéticaRESUMEN
Aging is an inevitable process of life. Defined by progressive physiological and functional loss of tissues and organs, aging increases the risk of mortality for the organism. The aging process is affected by various factors, including genetic and epigenetic ones. Here, we review the chromatin-specific epigenetic changes that occur during normal (chronological) aging and in premature aging diseases. Taking advantage of the reversible nature of epigenetic modifications, we will also discuss possible lifespan expansion strategies through epigenetic modulation, which was considered irreversible until recently.
Asunto(s)
Envejecimiento/genética , Epigénesis Genética , Epigenómica , Heterocromatina/genética , Factores de Edad , Animales , Cromatina/genética , Cromatina/metabolismo , Epigenómica/métodos , Regulación de la Expresión Génica , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , LongevidadRESUMEN
Cockayne syndrome is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and consumes and depletes cellular nicotinamide adenine dinucleotide, which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites, depletion of heterochromatin and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction.