RESUMEN

DNA repair proteins became the popular targets in research on cancer treatment. In our studies we hypothesized that inhibition of DNA polymerase theta (Polθ) and its combination with Poly (ADP-ribose) polymerase 1 (PARP1) or RAD52 inhibition and the alkylating drug temozolomide (TMZ) has an anticancer effect on glioblastoma cells (GBM21), whereas it has a low impact on normal human astrocytes (NHA). The effect of the compounds was assessed by analysis of cell viability, apoptosis, proliferation, DNA damage and cell cycle distribution, as well as gene expression. The main results show that Polθ inhibition causes a significant decrease in glioblastoma cell viability. It induces apoptosis, which is accompanied by a reduction in cell proliferation and DNA damage. Moreover, the effect was stronger when dual inhibition of Polθ with PARP1 or RAD52 was applied, and it is further enhanced by addition of TMZ. The impact on normal cells is much lower, especially when considering cell viability and DNA damage. In conclusion, we would like to highlight that Polθ inhibition used in combination with PARP1 or RAD52 inhibition has great potential to kill glioblastoma cells, and shows a synthetic lethal effect, while sparing normal astrocytes.

Asunto(s)

Supervivencia Celular , Glioblastoma , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína Recombinante y Reparadora de ADN Rad52 , Temozolomida , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Glioblastoma/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Línea Celular Tumoral , Temozolomida/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Supervivencia Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Polimerasa theta , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Mutaciones Letales Sintéticas/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo

RESUMEN

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.

RESUMEN

Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.

Asunto(s)

Unión Proteica , Proteína Recombinante y Reparadora de ADN Rad52 , Proteína de Replicación A , Dispersión del Ángulo Pequeño , Humanos , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/química , Proteína de Replicación A/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/genética , Recombinasa Rad51/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/genética , Difracción de Rayos X/métodos , Reparación del ADN , Modelos Moleculares , Dominios Proteicos

RESUMEN

DNA double-strand break (DSB) repair is a fundamental cellular process crucial for maintaining genome stability, with homologous recombination and non-homologous end joining as the primary mechanisms, and various alternative pathways such as single-strand annealing (SSA) and microhomology-mediated end joining also playing significant roles under specific conditions. IRC genes were previously identified as part of a group of genes associated with increased levels of Rad52 foci in Saccharomyces cerevisiae. In this study, we investigated the effects of IRC gene mutations on DSB repair, focusing on uncharacterized IRC10, 19, 21, 22, 23, and 24. Gene conversion (GC) assay revealed that irc10Δ, 22Δ, 23Δ, and 24Δ mutants displayed modest increases in GC frequencies, while irc19Δ and irc21Δ mutants exhibited significant reductions. Further investigation revealed that deletion mutations in URA3 were not generated in irc19Δ mutant cells following HO-induced DSBs. Additionally, irc19Δ significantly reduced frequency of SSA, and a synergistic interaction between irc19Δ and rad52Δ was observed in DSB repair via SSA. Assays to determine the choice of DSB repair pathways indicated that Irc19 is necessary for generating both GC and deletion products. Overall, these results suggest a potential role of Irc19 in DSB repair pathways, particularly in end resection process.

RESUMEN

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether-and if so, how-SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

Asunto(s)

Reparación del ADN , Proteína de la Leucemia Promielocítica , Sumoilación , Homeostasis del Telómero , Telómero , Humanos , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Telómero/metabolismo , Línea Celular Tumoral , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Línea Celular , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética

RESUMEN

Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.

Asunto(s)

Ciclina E , Inestabilidad Genómica , Mitosis , Proteínas Oncogénicas , Proteína Recombinante y Reparadora de ADN Rad52 , Humanos , Ciclina E/metabolismo , Ciclina E/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/genética , Replicación del ADN , Línea Celular Tumoral , Daño del ADN , ADN/metabolismo , ADN/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología

RESUMEN

Human centromeres are located within α-satellite arrays and evolve rapidly, which can lead to individual variation in array length. Proposed mechanisms for such alterations in length are unequal crossover between sister chromatids, gene conversion, and break-induced replication. However, the underlying molecular mechanisms responsible for the massive, complex, and homogeneous organization of centromeric arrays have not been experimentally validated. Here, we use droplet digital PCR assays to demonstrate that centromeric arrays can expand and contract within ∼20 somatic cell divisions of an alternative lengthening of telomere (ALT)-positive cell line. We find that the frequency of array variation among single-cell-derived subclones ranges from a minimum of ∼7% to a maximum of ∼100%. Further clonal evolution revealed that centromere expansion is favored over contraction. We find that the homologous recombination protein RAD52 and the helicase PIF1 are required for extensive array change, suggesting that centromere sequence evolution can occur via break-induced replication.

Asunto(s)

Centrómero , ADN Satélite , Humanos , Línea Celular , ADN Helicasas/genética

RESUMEN

BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation by at least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Femenino , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Reparación del ADN/genética , Genes BRCA2 , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Prueba de Complementación Genética

RESUMEN

Several sources of DNA damage compromise the integrity and stability of the genome of every organism. Specifically, DNA double-strand breaks (DSBs) can have lethal consequences for the cell. To repair this type of DNA damage, the cells employ homology-directed repair pathways or non-homologous end joining. Homology-directed repair requires the activity of the RAD52 epistasis group of genes. Rad52 is the main recombination protein in the budding yeast Saccharomyces cerevisiae, and rad52Δ mutants have been characterized to show severe defects in DSB repair and other recombination events. Here, we identified the RAD52 gene in the budding yeast Naumovozyma castellii. Our analysis showed that the primary amino acid sequence of N. castellii Rad52 shared 70% similarity with S. cerevisiae Rad52. To characterize the gene function, we developed rad52Δ mutant strains by targeted gene replacement transformation. We found that N. castellii rad52Δ mutants showed lowered growth capacity, a moderately altered cell morphology and increased sensitivity to genotoxic agents. The decreased viability of the N. castellii rad52Δ mutants in the presence of genotoxic agents indicates that the role of the Rad52 protein in the repair of DNA damage is conserved in this species.

Asunto(s)

Proteína Recombinante y Reparadora de ADN Rad52 , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Genoma Fúngico , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo

RESUMEN

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MX's ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MX's cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MX's cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting.

Asunto(s)

Mitoxantrona , Humanos , Masculino , ADN-Topoisomerasas de Tipo II , Proteínas de Unión al ADN , Mitoxantrona/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Proteoma , Proteómica , Sondas Moleculares/química , Sondas Moleculares/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Femenino

RESUMEN

Cells have evolved an arsenal of molecular mechanisms to respond to continuous alterations in the primary structure of DNA. At the cellular level, DNA damage response proteins accumulate at sites of DNA damage and organize into nuclear foci. As recounted by Errol Friedberg, pioneering work on DNA repair in the 1930 s was stimulated by collaborations between physicists and geneticists. In recent years, the introduction of ideas from physics on self-organizing compartments has taken the field of cell biology by storm. Percolation and phase separation theories are increasingly used to model the self-assembly of compartments, called biomolecular condensates, that selectively concentrate molecules without a surrounding membrane. In this review, we discuss these concepts in the context of the DNA damage response. We discuss how studies of DNA repair foci as condensates can link molecular mechanisms with cell physiological functions, provide new insights into regulatory mechanisms, and open new perspectives for targeting DNA damage responses for therapeutic purposes.

Asunto(s)

Núcleo Celular , Proteínas , Proteínas/química , Daño del ADN , Reparación del ADN

RESUMEN

The breast cancer susceptibility gene 1/2 (BRCA1/2) are the key regulators in maintaining the genomic integrity and mutations in these genes have been associated with development of breast and ovarian cancers. Also, synthetic lethality has been shown in BRCA1/2 deficient cancers, when the RAD52 gene is silenced by shRNA or small molecules aptamers, suggesting a role for RAD52 in the breast cancers pathogenesis. Thus, to find the potential inhibitors of RAD52, a collection of 21,000 compounds from the ChemBridge screening library was screened to conduct molecular docking and molecular dynamics simulation (MD) against RAD52. Further, the results were validated by a density functional theory (DFT) analysis and using post-dynamics free energy calculations. Out of all screened molecules, the docking study revealed five compounds were found to have promising activities against RAD52. Moreover, the catalytic amino acid residues of RAD52 developed stable contacts with compound 8758 and 10593, as anticipated by DFT calculation, MD simulation, and post dynamics MM-GBSA energy calculation. It appears that compound 8758 is the best inhibitor against RAD52 followed by 10593 compared to the other top hits, in terms of the HOMO orbital energy (-1.0966 eV and -1.2136 eV) from DFT and the post dynamics binding free energy calculation (-54.71 and -52.43 Kcal/mol). Furthermore, a drug-like properties of lead molecules (8758 and 10593) were also seen via ADMET analysis. Based on our computational analysis, we hypothesize that a small molecule 8758 and 10593 possess the therapeutic potential in the management for breast cancer patients with a BRCA mutation via targeting RAD52.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Carcinoma de Células Renales , Neoplasias Renales , Humanos , Pacientes , Pronóstico , Biomarcadores de Tumor , Proteína Recombinante y Reparadora de ADN Rad52

RESUMEN

In recent years, the RAD52 protein has been highlighted as a mediator of many DNA repair mechanisms. While RAD52 was initially considered to be a non-essential auxiliary factor, its inhibition has more recently been demonstrated to be synthetically lethal in cancer cells bearing mutations and inactivation of specific intracellular pathways, such as homologous recombination. RAD52 is now recognized as a novel and critical pharmacological target. In this review, we comprehensively describe the available structural and functional information on RAD52. The review highlights the pathways in which RAD52 is involved and the approaches to RAD52 inhibition. We discuss the multifaceted role of this protein, which has a complex, dynamic, and functional 3D superstructural arrangement. This complexity reinforces the need to further investigate and characterize RAD52 to solve a challenging mechanistic puzzle and pave the way for a robust drug discovery campaign.

RESUMEN

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Cisplatino/uso terapéutico , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Gemcitabina/uso terapéutico , Neoplasias Pulmonares/genética , Proteína Recombinante y Reparadora de ADN Rad52 , Factores de Transcripción

RESUMEN

G-quadruplex (G4)-forming DNA sequences are abundant in the human genome, and they are hot spots for inducing DNA double-strand breaks (DSBs) and genome instability. The mechanisms involved in protecting G4s and maintaining genome stability have not been fully elucidated. Here, we demonstrated that RAD52 plays an important role in suppressing DSB accumulation at G4s, and RAD52-deficient cells are sensitive to G4-stabilizing compounds. Mechanistically, we showed that RAD52 is required for efficient homologous recombination repair at G4s, likely due to its function in recruiting structure-specific endonuclease XPF to remove G4 structures at DSB ends. We also demonstrated that upon G4 stabilization, endonuclease MUS81 mediates cleavage of stalled replication forks at G4s. The resulting DSBs recruit RAD52 and XPF to G4s for processing DSB ends to facilitate homologous recombination repair. Loss of RAD52 along with G4-resolving helicase FANCJ leads to a significant increase of DSB accumulation before and after treatment with the G4-stabilizing compound pyridostatin, and RAD52 exhibits a synthetic lethal interaction with FANCJ. Collectively, our findings reveal a new role of RAD52 in protecting G4 integrity and provide insights for new cancer treatment strategies.

Asunto(s)

G-Cuádruplex , Proteína Recombinante y Reparadora de ADN Rad52 , Animales , Humanos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Endonucleasas/metabolismo , Inestabilidad Genómica , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Reparación del ADN por Recombinación/genética

RESUMEN

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Asunto(s)

Proteína BRCA2 , Neoplasias Ováricas , Humanos , Femenino , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína BRCA1/metabolismo , Reparación del ADN , Neoplasias Ováricas/genética , Descubrimiento de Drogas , Relación Estructura-Actividad

RESUMEN

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.

Asunto(s)

ARN Largo no Codificante , Telomerasa , Homeostasis del Telómero , Telómero/genética , Telómero/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Estructuras R-Loop/genética , Reparación del ADN

RESUMEN

O-Linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.

Asunto(s)

Acetilglucosamina , Rotura Cromosómica , Acetilglucosamina/metabolismo , ADN , Humanos , N-Acetilglucosaminiltransferasas , Serina/metabolismo , Treonina/metabolismo

RESUMEN

Nitroglycerin (NTG)-a nitric oxide-donating drug-is traditionally administered via the sublingual route to treat acute myocardial angina attacks. NTG also increases tumor blood flow and, consequently, cancer drug delivery to tumor cells. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating radiation-sensitive 51 (Rad51) recombinase activity. Pemetrexed-a multitargeted antifolate agent-exhibits satisfactory clinical activity in wild-type nonsquamous non-small-cell lung cancer (NSCLC) cells. However, the synergistic activity of combination therapy with NTG and pemetrexed against NSCLC cells has not yet been clarified. In 2 NSCLC cell lines (i.e. lung squamous cell carcinoma H520 and lung adenocarcinoma H1975 cells), NTG reduced Rad52 expression; in addition, decreased phospho-AKT and phospho-ERK1/2 protein levels were observed. Enhancement of AKT or ERK1/2 activity through transfection with a constitutively active AKT (AKT-CA) vector or constitutively active mitogen-activated protein kinase kinase 1 (MKK1-CA) vector increased the Rad52 protein level and cell survival, which were suppressed by NTG. The knockdown of Rad52 expression by using small interfering RNA or by inhibiting AKT and ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition induced by NTG. Moreover, NTG synergistically enhanced the cytotoxicity and cell growth inhibition induced by pemetrexed in NSCLC cells; these effects were associated with AKT and ERK1/2 inactivation and, consequently, Rad52 downregulation in H520 and H1975 cells. The results provide a rationale for combining NTG and pemetrexed in lung cancer treatment to improve lung cancer control.