RESUMEN
UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5' to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post-translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X-ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal-coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.
Asunto(s)
Reparación del ADN/genética , Endonucleasas/química , Endonucleasas/genética , Lisina/metabolismo , Thermus thermophilus/enzimología , Sitios de Unión/genética , Cristalografía por Rayos X , ADN/metabolismo , Endonucleasas/metabolismo , Modelos Moleculares , Mutación Puntual/genética , Procesamiento Proteico-Postraduccional/genética , Rayos UltravioletaRESUMEN
UV damage endonuclease (UVDE) from Schizosaccharomyces pombe initiates repair of UV lesions and abasic sites by nicking the DNA 5' to the damaged site. In this paper we show that in addition UVDE incises DNA containing a single-strand nick or gap, but that the enzymatic activity on these substrates as well as on abasic sites strongly depends on the presence of a neighbouring pyrimidine residue. This indicates that, although UVDE may have been derived from an ancestral AP endonuclease its major substrate is a UV lesion and not an AP site. We propose that UVDE rotates two nucleotides into a pocket of the protein in order to bring the scissile bond close to the active site and that purine bases are excluded from this pocket. We also show that in the DNA complex residue Tyr-358 of UVDE penetrates the DNA helix causing unstacking of two residues opposite the lesion, thereby stabilizing the protein-DNA interaction, most likely by promoting bending of the DNA. In the absence of Tyr-358 the enzyme exhibits an increased catalytic activity on UV-induced lesions, but only at a lower pH of 6.5. At physiological conditions (pH 7.5) the mutant protein completely looses its catalytic activity although it can still bind to the DNA. We propose that in addition to stabilizing the bend in the DNA the hydrophobic side chain of Tyr-358 shields the active site from exposure to the solvent.
Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN/efectos de la radiación , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , 2-Aminopurina/metabolismo , Western Blotting , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Reparación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Mutación/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Tirosina/genética , Tirosina/metabolismo , Rayos UltravioletaRESUMEN
The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.