RESUMEN
BACKGROUND: DNA double-strand breaks (DSBs) can occur in response to ionizing radiation (IR), radiomimetic agents and from endogenous DNA-damaging reactive oxygen metabolites. Unrepaired or improperly repaired DSBs are potentially the most lethal form of DNA damage and can result in chromosomal translocations and contribute to the development of cancer. The principal mechanism for the repair of DSBs in humans is non-homologous end-joining (NHEJ). Ku is a key member of the NHEJ pathway and plays an important role in the recognition step when it binds to free DNA termini. Ku then stimulates the assembly and activation of other NHEJ components. DNA binding of Ku is regulated by redox conditions and evidence from our laboratory has demonstrated that Ku undergoes structural changes when oxidized that results in a reduction in DNA binding activity. The C-terminal domain and cysteine 493 of Ku80 were investigated for their contribution to redox regulation of Ku. RESULTS: We effectively removed the C-terminal domain of Ku80 generating a truncation mutant and co-expressed this variant with wild type Ku70 in an insect cell system to create a Ku70/80DeltaC heterodimer. We also generated two single amino acid variants of Cys493, replacing this amino acid with either an alanine (C493A) or a serine (C493S), and over-expressed the variant proteins in SF9 insect cells in complex with wild type Ku70. Neither the truncation nor the amino acid substitutions alters protein expression or stability as determined by SDS-PAGE and Western blot analysis. We show that the C493 mutations do not alter the ability of Ku to bind duplex DNA in vitro under reduced conditions while truncation of the Ku80 C-terminus slightly reduced DNA binding affinity. Diamide oxidation of cysteines was shown to inhibit DNA binding similarly for both the wild-type and all variant proteins. Interestingly, differential DNA binding activity following re-reduction was observed for the Ku70/80DeltaC truncation mutant. CONCLUSION: Together, these results suggest that the C-terminal domain and C493 of Ku80 play at most a minor role in the redox regulation of Ku, and that other cysteines are likely involved, either alone or in conjunction with these regions of Ku80.
Asunto(s)
Antígenos Nucleares/química , Antígenos Nucleares/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Animales , Antígenos Nucleares/genética , Línea Celular , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Insectos , Autoantígeno Ku , Mutación , Oxidación-Reducción , Estructura Terciaria de ProteínaRESUMEN
The Saccharomyces cerevisiae mutant strain YW778, which lacks apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase DNA repair activities, displays high levels of spontaneous mutations and hypersensitivities to several DNA damaging agents. We searched a cDNA library derived from the nematode Caenorhabditis elegans for gene products that would rescue the DNA repair defects of this yeast mutant. We isolated two genes, apn-1 and exo-3, encoding proteins that have not been previously characterized. Both APN-1 and EXO-3 share significant identity with the functionally established Escherichia coli AP endonucleases, endonuclease IV and exonuclease III, respectively. Strain YW778 expressing either apn-1 or exo-3 shows parental levels of spontaneous mutations, as well as resistance to DNA damaging agents that produce AP sites and DNA single strand breaks with blocked 3'-ends. Using an in vitro assay, we show that the apn-1 and exo-3 genes independently express AP endonuclease activity in the yeast mutant. We further characterize the EXO-3 protein and three of its mutated variants E68A, D190A, and H279A. The E68A variant retains both AP endonuclease and 3'-diesterase repair activities in vitro, yet severely lacks the ability to protect strain YW778 from spontaneous and drug-induced DNA lesions, suggesting that this variant E68A may possess a defect that interferes with the repair process in vivo. In contrast, D190A and H279A are completely devoid of DNA repair activities and fail to rescue the genetic instability of strain YW778. Our data strongly suggest that EXO-3 and APN-1 are enzymes possessing intrinsic AP endonuclease and 3'-diesterase activities.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Prueba de Complementación Genética , Proteínas del Helminto/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/aislamiento & purificación , Reparación del ADN , ADN Bacteriano , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/aislamiento & purificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/aislamiento & purificación , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Expresión Génica , Biblioteca de Genes , Vectores Genéticos , Glutatión Transferasa/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Insercional , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The Caenorhabditis elegans genes, exo-3 and apn-1, encode the proteins EXO-3 and APN-1, belonging to the exo III and endo IV families of apurinic/apyrimidinic (AP) endonucleases/3'-diesterases, respectively. Homologues of EXO-3 and APN-1 in E. coli and yeast have been clearly documented to repair AP sites and DNA strand breaks with blocked 3' ends to prevent genomic instability. Herein, we purified the C. elegans EXO-3, expressed as a Gst-fusion protein in yeast, and demonstrated that it possesses strong AP endonuclease and 3'-diesterase activities. However, unlike the E. coli counterpart exonuclease III, EXO-3 shows no significant level of 3' --> 5' exonuclease activity following incision at AP sites. In addition, EXO-3 lacks the ability to directly incise DNA at the 5' side of various oxidatively damaged bases, as observed for the human counterpart Ape1, suggesting that C. elegans evolved a member with tailored functions. Importantly, a variant form of EXO-3, E68A, demonstrates altered magnesium-binding properties, and although the in vitro AP endonuclease is nearly fully recovered in the presence of MgCl2, the 3'-diesterase activity is reduced when compared to the native enzyme. We suggest that Glu68 plays a role in coordinating Mg2+ binding for the enzyme catalytic mechanism. Further analysis reveals that neither purified Gst-EXO-3 nor the E68A variant forms a readily detectable DNA-protein complex with an oligonucleotide substrate containing either an AP site or an alpha,beta-unsaturated aldehyde at its 3' end. However, if the reaction is conducted in the presence of crude extracts derived from either yeast or C. elegans embryos, only E68A forms a distinct slow migrating DNA-protein complex with each of the substrates, suggesting that Glu68 may be required to facilitate the release of EXO-3 from the incised DNA to allow entry of the remaining components of the base-excision repair pathway. Thus, the slow migrating DNA-protein complex formed by the E68A variant could be indicative of a stalled repair process with associated factor(s).
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Exodesoxirribonucleasas/metabolismo , Magnesio/química , Aldehídos/química , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination. The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic. Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans. Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C. elegans. These enzyme activities were monitored using a defined 5'-end (32)P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21. The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product. The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed. Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase. The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5'-cleavage of the AP site. Further analysis revealed that product formation was dependent upon the presence of Mg(2+), suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg(2+)-dependent AP endonuclease. It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C. elegans.