RESUMEN
Topoisomerase 1 (Top1) reversibly nicks chromosomal DNA to relax strain accumulated during transcription, replication, chromatin assembly, and chromosome condensation. The Top1 poison camptothecin targets cancer cells by trapping the enzyme in the covalent complex Top1cc, tethered to cleaved DNA by a tyrosine-3'-phosphate bond. In vitro mechanistic studies point to interfacial inhibition, where camptothecin binding to the Top1-DNA interface stabilizes Top1cc. Here we present a complementary covalent mechanism that is critical in vivo. We observed that camptothecins induce oxidative stress, leading to lipid peroxidation, lipid-derived electrophile accumulation, and Top1 poisoning via covalent modification. The electrophile 4-hydroxy-2-nonenal can induce Top1cc on its own and forms a Michael adduct to a cysteine thiol in the Top1 active site, potentially blocking tyrosine dephosphorylation and 3' DNA phosphate release. Thereby, camptothecins may leverage a physiological cysteine-based redox switch in Top1 to mediate their selective toxicity to rapidly proliferating cancer cells.
Asunto(s)
Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Lípidos/química , Camptotecina/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Estructura Molecular , Estrés Oxidativo/efectos de los fármacosRESUMEN
DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.
Asunto(s)
Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Autoantígeno Ku/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Células Cultivadas , ADN/química , Roturas del ADN de Doble Cadena , Edición Génica , Humanos , Autoantígeno Ku/química , Ratones , Inhibidores de Proteínas Quinasas/químicaRESUMEN
DNA topoisomerases are essential for DNA metabolic processes such as replication and transcription. Since DNA is double stranded, the unwinding needed for these processes results in DNA supercoiling and catenation of replicated molecules. Changing the topology of DNA molecules to relieve supercoiling or resolve catenanes requires that DNA be transiently cut. While topoisomerases carry out these processes in ways that minimize the likelihood of genome instability, there are several ways that topoisomerases may fail. Topoisomerases can be induced to fail by therapeutic small molecules such as by fluoroquinolones that target bacterial topoisomerases, or a variety of anti-cancer agents that target the eukaryotic enzymes. Increasingly, there have been a large number of agents and processes, including natural products and their metabolites, DNA damage, and the intrinsic properties of the enzymes that can lead to long-lasting DNA breaks that subsequently lead to genome instability, cancer, and other diseases. Understanding the processes that can interfere with topoisomerases and how cells respond when topoisomerases fail will be important in minimizing the consequences when enzymes need to transiently interfere with DNA integrity.
Asunto(s)
Daño del ADN , ADN-Topoisomerasas/metabolismo , Productos Biológicos/farmacología , Reparación del ADN/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Humanos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3' and 5' terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Fosforilación , Dominios y Motivos de Interacción de ProteínasRESUMEN
The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Hidrolasas Diéster Fosfóricas/metabolismo , Antígenos Nucleares/metabolismo , ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Autoantígeno KuRESUMEN
The recent discovery of numerous human short open reading frame (sORF)-encoded polypeptides (SEPs) has raised important questions about the functional roles of these molecules in cells. Here, we show that a 69-amino acid SEP, MRI-2, physically interacts with the Ku heterodimer to stimulate DNA double-strand break ligation via nonhomologous end joining. The characterization of MRI-2 suggests that this SEP may participate in DNA repair and underscores the potential of SEPs to serve important biological functions in mammalian cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , ADN Helicasas/metabolismo , Sistemas de Lectura Abierta/fisiología , Línea Celular , ADN Helicasas/genética , Humanos , Autoantígeno KuRESUMEN
In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.