RESUMEN
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Asunto(s)
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidad por Sustrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/químicaRESUMEN
Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , Endonucleasas/metabolismo , Endonucleasas/genéticaRESUMEN
Hydrolytic ring opening of guanine N7-adducts with compounds containing an oxacyclopropane ring, namely glycidamide, glycidol and 1,2-epoxybutane, was analyzed, and the reaction of the glycidamide adduct was the fastest. The differences in the reaction rates were confirmed by theoretical calculations.
RESUMEN
The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.
Asunto(s)
Daño del ADN , Desoxirribonucleasa I , Rayos Ultravioleta , Rayos Ultravioleta/efectos adversos , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasa I/química , ADN/metabolismo , ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Bacteriófago T7/enzimología , Bacteriófago T7/genética , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/química , Reparación del ADNRESUMEN
Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.
Asunto(s)
Aductos de ADN , Mutágenos , Humanos , Acrilamidas , Desoxiguanosina , ADN , Daño del ADN , Replicación del ADN , Mutagénesis , Mutágenos/toxicidad , Contaminación de AlimentosRESUMEN
Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.
Asunto(s)
Proteínas de Unión al ADN , Magnesio , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Magnesio/metabolismo , Nucleósidos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Daño del ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores de Topoisomerasa , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN , Reparación del ADNRESUMEN
Apurinic/apyrimidinic (AP) sites are DNA lesions created under normal growth conditions that result in cytotoxicity, replication-blocks, and mutations. AP sites are susceptible to ß-elimination and are liable to be converted to DNA strand breaks. HMCES (5-hydroxymethylcytosine binding, ES cell specific) protein interacts with AP sites in single stranded (ss) DNA exposed at DNA replication forks to generate a stable thiazolidine protein-DNA crosslink and protect cells against AP site toxicity. The crosslinked HMCES is resolved by proteasome-mediated degradation; however, it is unclear how HMCES-crosslinked ssDNA and the resulting proteasome-degraded HMCES adducts are processed and repaired. Here, we describe methods for the preparation of thiazolidine adduct-containing oligonucleotides and determination of their structure. We demonstrate that the HMCES-crosslink is a strong replication blocking adduct and that protease-digested HMCES adducts block DNA replication to a similar extent as AP sites. Moreover, we show that the human AP endonuclease APE1 incises DNA 5' to the protease-digested HMCES adduct. Interestingly, while HMCES-ssDNA crosslinks are stable, the crosslink is reversed upon the formation of dsDNA, possibly due to a catalytic reverse reaction. Our results shed new light on damage tolerance and repair pathways for HMCES-DNA crosslinks in human cells.
Asunto(s)
Aductos de ADN , Reparación del ADN , Humanos , Tiazolidinas , Complejo de la Endopetidasa Proteasomal/metabolismo , ADN/química , Daño del ADN , ADN de Cadena Simple/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismoRESUMEN
Mismatch repair (MMR) is a highly conserved DNA repair pathway that corrects mismatched bases during DNA replication. The biological significance of MMR in human cells is underscored by the fact that dysfunction of the MMR pathway results in Lynch syndrome, which is associated with a genetic predisposition to different cancer types. We have previously established a reporter mismatch plasmid to evaluate MMR using fluorescent proteins in living cells. However, the preparation of these plasmids requires significant amounts of time and money, which reduces their broad applicability. To overcome the abovementioned limitations, we produced in this study a novel reporter plasmid, pBSII NLS-MC-EGFP-tdTomato (pBET2), that can be used in the oligo swapping method. In this method, a nicking endonuclease produces a single-stranded DNA gap on a double-stranded DNA plasmid that can be replaced by ligation with synthetic oligonucleotides. It is significantly easier and more user-friendly than previous assays, which require in vitro DNA synthesis with single-stranded plasmid DNA and purification using ultracentrifugation in cesium chloride-ethidium bromide gradients. The plasmid also contains a nicking site that allows the MMR repair machinery to efficiently distinguish the newly synthesized strand as a target for repair. In addition, a nuclear localization signal facilitates green fluorescent protein expression in the nucleus, which helps to verify the effectiveness of MMR using fluorescence microscopy. Similar to the previous reporter plasmid, this construct facilitates the assessment of MMR proficiency in human living cells via the expression of fluorescent proteins while overcoming many of the negative aspects of the previous protocol.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Reparación del ADN , ADN/metabolismo , Fluorescencia , Humanos , Plásmidos/genéticaRESUMEN
(6-4) Photolyases ((6-4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6-4)pyrimidone photoproducts. To repair DNA, (6-4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH- via a chain of three or four conserved tryptophan residues, transiently forming FADâ¢-/FADH- ⯠TrpHâ¢+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor-acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpHâ¢+ or protonation of FADâ¢-. Here, we address Arabidopsis thaliana (6-4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins-due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3Hâ¢+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3Hâ¢+ stabilization.
Asunto(s)
Arabidopsis , Desoxirribodipirimidina Fotoliasa , Arabidopsis/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Transporte de Electrón , Electrones , Flavina-Adenina Dinucleótido/metabolismo , Oxidación-Reducción , Triptófano/metabolismoRESUMEN
Human mitochondrial DNA contains more UV-induced lesions than the nuclear DNA due to lack of mechanism to remove bulky photoproducts. Human DNA polymerase gamma (Pol γ) is the sole DNA replicase in mitochondria, which contains a polymerase (pol) and an exonuclease (exo) active site. Previous studies showed that Pol γ only displays UV lesion bypassing when its exonuclease activity is obliterated. To investigate the reaction environment on Pol γ translesion activity, we tested Pol γ DNA activity in the presence of different metal ions. While Pol γ is unable to replicate through UV lesions on DNA templates in the presence of Mg2+, it exhibits robust translesion DNA synthesis (TLS) on cyclobutane pyrimidine dimer (CPD)-containing template when Mg2+ was mixed with or completely replaced by Mn2+. Under these conditions, the efficiency of Pol γ's TLS opposite CPD is near to that on a non-damaged template and is 800-fold higher than that of exonuclease-deficient Pol γ. Interestingly, Pol γ exhibits higher exonuclease activity in the presence of Mn2+ than with Mg2+, suggesting Mn2+-stimulated Pol γ TLS is not via suppressing its exonuclease activity. We suggest that Mn2+ ion expands Pol γ's pol active site relative to Mg2+ so that a UV lesion can be accommodated and blocks the communication between pol and exo active sites to execute translesion DNA synthesis.
RESUMEN
The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.
Asunto(s)
Reparación del ADN , Replicación del ADN , Daño del ADN , Reparación del ADN/genética , Replicación del ADN/genética , Mutagénesis , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA that is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in the regulation of inosine-containing RNA.
Asunto(s)
Desoxirribonucleasa (Dímero de Pirimidina) , Proteínas ViralesRESUMEN
Nucleotide excision repair (NER) is a repair mechanism that removes DNA lesions induced by UV radiation, environmental mutagens and carcinogens. There exists sufficient evidence against acetaldehyde suggesting it to cause a variety of DNA lesions and be carcinogenic to humans. Previously, we found that acetaldehyde induces reversible intra-strand GG crosslinks in DNA similar to those induced by cis-diammineplatinum(II) that is subsequently repaired by NER. In this study, we analysed the repairability by NER mechanism and the mutagenesis of acetaldehyde. In an in vitro reaction setup with NER-proficient and NER-deficient xeroderma pigmentosum group A (XPA) cell extracts, NER reactions were observed in the presence of XPA recombinant proteins in acetaldehyde-treated plasmids. Using an in vivo assay with living XPA cells and XPA-correcting XPA cells, the repair reactions were also observed. Additionally, it was observed that DNA polymerase eta inserted dATP opposite guanine in acetaldehyde-treated oligonucleotides, suggesting that acetaldehyde-induced GG-to-TT transversions. These findings show that acetaldehyde induces NER repairable mutagenic DNA lesions.
Asunto(s)
Acetaldehído/efectos adversos , Reparación del ADN/efectos de los fármacos , ADN/genética , Mutagénesis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , Fibroblastos/efectos de los fármacos , Humanos , Mutagénesis/genética , Mutágenos/efectos adversos , Transfección/métodos , Rayos Ultravioleta , Xerodermia Pigmentosa/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/genéticaRESUMEN
Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.
Asunto(s)
Arabidopsis/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Inosina/química , ARN de Planta/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Desoxirribonucleasa (Dímero de Pirimidina)/genética , Regulación de la Expresión Génica de las Plantas , Edición de ARN , ARN de Planta/química , Especificidad por SustratoRESUMEN
Photolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6-4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6-4) photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6-4) photolyase by spectroscopic studies.
Asunto(s)
Desoxirribodipirimidina Fotoliasa/química , Riboflavina/análogos & derivados , Animales , Desoxirribodipirimidina Fotoliasa/metabolismo , Transferencia de Energía , Riboflavina/química , Riboflavina/metabolismo , Xenopus laevisRESUMEN
Photolyases are flavoenzymes responsible for the repair of carcinogenic DNA damage caused by ultraviolet radiation. They harbor the catalytic cofactor flavin adenine dinucleotide (FAD). The light-driven electron transfer from the excited state of the fully-reduced form of FAD to the DNA lesions causes rearrangement of the covalent bonds, leading to the restoration of intact nucleobases. In addition to the catalytic chromophore, some photolyases bear a secondary chromophore with better light absorption capability than FAD, acting as a light-harvesting chromophore that harvests photons in sunlight efficiently and transfers light energy to the catalytic center, as observed in natural photoreceptor proteins. Inspired by nature, we covalently and site-specifically attached a synthetic chromophore to the surface of photolyase using oligonucleotides containing a modified nucleoside and a cyclobutane-type DNA lesion, and successfully enhanced its enzymatic activity in the light-driven DNA repair. Peptide mapping in combination with theoretical calculations identified the amino acid residue that binds to the chromophore, working as an artificial light-harvesting chromophore. Our results broaden the strategies for protein engineering and provide a guideline for tuning of the light perception abilities and enzymatic activity of the photoreceptor proteins.
Asunto(s)
Reparación del ADN , Desoxirribodipirimidina Fotoliasa/metabolismo , Ingeniería de Proteínas , Daño del ADN , Flavina-Adenina Dinucleótido/metabolismo , Luz , Proteínas Recombinantes/metabolismo , Thermus thermophilus/enzimologíaRESUMEN
Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.
Asunto(s)
Roturas del ADN de Cadena Simple/efectos de la radiación , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Rayos Ultravioleta/efectos adversos , Sistemas CRISPR-Cas/genética , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Fibroblastos , Técnicas de Inactivación de Genes , Humanos , Células MCF-7 , Cultivo Primario de Células , Piel/citología , Piel/patología , Piel/efectos de la radiación , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Xerodermia Pigmentosa/etiología , Xerodermia Pigmentosa/patología , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
Endonuclease III (EndoIII) is nearly ubiquitous in all three domains of life. EndoIII family proteins exhibit a bifunctional (glycosylase/lyase) activity on oxidative/saturated pyrimidine bases, such as thymine glycol. Previous studies on EndoIII homologs have reported the presence of important residues involved in substrate binding and catalytic activity. However, a biochemical clarification of the roles of these residues as well as details of their evolutionary conservation is still lacking. This is particularly true for archaeal orthologs. The current study demonstrated the roles of the evolutionarily conserved residues of euryarchaeon Thermococcus kodakarensis EndoIII (TkoEndoIII). We utilized amino acid sequence analysis and homology modeling to identify highly conserved regions with potential key residues in the EndoIII proteins. Using Ala-substituted TkoEndoIII mutant proteins, residues of interest were quantitatively examined via DNA binding, glycosylase/AP lyase/bifunctional activity, and DNA trapping assays. The obtained results allowed us to determine the roles, as well as the significance of these roles in Schiff base formation (Lys140 as a nucleophile and Asp158), Tg recognition (His160), substrate binding (Arg59, Leu101, Trp102, and Gly136), ß-elimination activities (Ser57 and Asp62), and [4Fe-4S] cluster formation (Cys208 and Cys215). Interestingly, a critical role played by the highly conserved Lys105 (predicted as being away from the catalytic site) in substrate binding, accompanied by a significant indirect effect on catalytic activity, were detected. Our results suggest that these particular residues play conserved roles among EndoIII orthologs across the domains. In addition to identifying the critical role of the highly conserved Lys105, the study provides a comprehensive understanding of the functions attributable to the evolutionarily conserved residues found in the EndoIII family, from Escherichia coli to humans.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Elementos Estructurales de las Proteínas , Thermococcus/enzimología , Timina/análogos & derivados , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , ADN Glicosilasas/química , ADN Glicosilasas/genética , Reparación del ADN , ADN de Archaea/metabolismo , Evolución Molecular , Cinética , Análisis de Secuencia de Proteína , Especificidad por Sustrato , Thermococcus/genética , Timina/metabolismoRESUMEN
Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.