RESUMO
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.
Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Transferases/metabolismo , Alquilação , Células Cultivadas , Adutos de DNA/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Células PC-3 , Ligação Proteica , Rad51 Recombinase/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Mycobacterium leprae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano/efeitos dos fármacos , Humanos , Hanseníase/microbiologia , Modelos Moleculares , Mycobacterium leprae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
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.