Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

Emily C Beckwitt,Sunbok Jang,Nina Simon,Jochen Kuper,Isadora Carnaval Detweiler,Caroline Kisker,Thomas Carell,Johanna Bretzler,Florian Sauer,Bennett Van Houten,Simon C Watkins

doi:10.1038/s41467-020-15168-1

Abstract

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.

Highlights

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UVinduced photoproducts and bulky base adducts
We first confirmed that XPA recognizes AAF-adducted DNA by electrophoretic mobility shift assay (EMSA) as reported by others[8,9,10,12]
It is important to note that any affinity of XPA for DNA ends could obscure EMSA results in terms of (a) specificity, as end-binding would increase overall binding on both substrates, thereby lowering the apparent specificity for the lesion, and (b) stoichiometry, as separate XPA proteins bound to the lesion and the end of the DNA would migrate the same as a true dimer in the gel

Summary

Introduction

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UVinduced photoproducts and bulky base adducts. Nucleotide excision repair (NER) is a highly conserved DNA repair pathway that, through a series of damage recognition and verification steps, is able to act on a wide range of DNA lesions In humans, this process involves approximately 30 proteins, working together to protect our genomes from the damaging effects of UV radiation and chemical agents like cisplatin, polycyclic aromatic hydrocarbons, and aromatic amines. It has been suggested that damage recognition in NER, which needs to accommodate a diverse range of structures, is accomplished via a “discrimination cascade” involving multiple proteins with imperfect selectivity[7,25,26] In support of this model, XPA enhances the damage specificity of TFIIH by promoting both its translocation along non-damaged DNA and stalling on damaged DNA20. XPA stoichiometry both on and off of DNA remains controversial as others have reported monomeric binding[15,21,36], or that stoichiometry is dependent on the DNA substrate[16]

Methods

Results

Conclusion