RESUMEN
Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, â¼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Daño del ADN/fisiología , Reparación del ADN/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293 , HumanosRESUMEN
The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Daño del ADN , ADN Ribosómico/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Nucléolo Celular/genética , ADN Ribosómico/genética , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosforilación/genética , Transcripción Genética/genéticaRESUMEN
DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smoke-induced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Genómica/métodos , Benzo(a)pireno/metabolismo , Aductos de ADN/metabolismo , Humanos , Plásmidos/genética , Homología de Secuencia , Rayos UltravioletaRESUMEN
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
Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.
Asunto(s)
Genoma Humano , Proteoma/análisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Pirimidinas/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability.
Asunto(s)
Daño del ADN , Reparación del ADN , Recombinación Genética , Animales , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Células Cultivadas , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Humanos , Ratones , Proteínas Nucleares/fisiología , Recombinasa Rad51/fisiologíaRESUMEN
Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.
Asunto(s)
Reparación del ADN , Escherichia coli/genética , Recombinación Genética/genética , Moldes Genéticos , Secuencia de Bases , Benzo(a)pireno/metabolismo , Aductos de ADN/metabolismo , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasa V/metabolismo , Guanina/metabolismo , Modelos Biológicos , Mutación/genética , Plásmidos , Rec A Recombinasas/metabolismoRESUMEN
Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR). Here we show that RR effectively repairs gaps opposite DNA lesions. When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events. This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR. Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR. This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.