RESUMEN

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.

Asunto(s)

ADN/química , ADN/metabolismo , Imagen Individual de Molécula/métodos , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Biofisica/métodos , Aductos de ADN/química , Aductos de ADN/metabolismo , Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Humanos , Microscopía de Fuerza Atómica , Unión Proteica , Rayos Ultravioleta