RESUMEN
DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Alquilantes/toxicidad , Daño del ADN , Reparación del ADN/fisiología , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Citosina/análogos & derivados , Citosina/metabolismo , Aductos de ADN , Metilación de ADN , Desmetilación , Células HEK293 , Humanos , Mesilatos/toxicidadRESUMEN
The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3'-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C-ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Transferasas/metabolismo , Alquilación , Células Cultivadas , Aductos de ADN/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Células PC-3 , Unión Proteica , Recombinasa Rad51/metabolismoRESUMEN
Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , Mycobacterium leprae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Alquilación/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano/efectos de los fármacos , Humanos , Lepra/microbiología , Modelos Moleculares , Mycobacterium leprae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/antagonistas & inhibidores , Indenos/farmacología , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Calorimetría , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Indenos/síntesis química , Indenos/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The clinical diagnosis of traumatic brain injury (TBI) is based on neurological examination and neuro-imaging tools such as CT scanning and MRI. However, neurological examination at times may be confounded by consumption of alcohol or drugs and neuroimaging facilities may not be available at all centers. Human ubiquitin C-terminal hydrolase (UCHL1) is a well-accepted serum biomarker for severe TBI and can be used to detect the severity of a head injury. A reliable, rapid, cost effective, bedside and easy to perform method for the detection of UCHL1 is a pre-requisite for wide clinical applications of UCHL1 as a TBI biomarker. We developed a rapid detection method for UCHL1 using surface plasmon resonance of gold nanoparticles with a limit of detection (LOD) of 0.5 ng mL-1. It has a sensitivity and specificity of 100% each and meets an analytical precision similar to that of conventional sandwich ELISA but can be performed rapidly. Using this method we successfully detected UCHL1 in a cohort of 66 patients with TBI and were reliably able to distinguish mild TBI from moderate to severe TBI.