RESUMO
Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.
Assuntos
Adenina/análogos & derivados , DNA Glicosilases , Reparo do DNA , Mutagênicos/farmacologia , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Adenina/farmacologia , Substituição de Aminoácidos , Animais , Domínio Catalítico , DNA Glicosilases/química , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Expressão Gênica , Instabilidade Genômica , Células HEK293 , Humanos , Cinética , Camundongos , Camundongos Knockout , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. RESULTS: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. CONCLUSION: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.
Assuntos
Embrião não Mamífero/enzimologia , Proteínas de Peixes/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Animais , Cálcio/metabolismo , Extratos Celulares/química , DNA/metabolismo , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/embriologia , Ativação Enzimática , Escherichia coli/genética , Proteínas de Peixes/isolamento & purificação , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Extratos de Tecidos/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Peixe-Zebra/embriologia , Zinco/metabolismoRESUMO
Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5'-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3'- and 5'-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3'- and 5'-phosphotyrosyl linkage at the 3' and 5' ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5'-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5' activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC(50)=40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.
