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

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Listed:
  • Emily C. Beckwitt

    (University of Pittsburgh
    UPMC Hillman Cancer Center)

  • Sunbok Jang

    (UPMC Hillman Cancer Center
    University of Pittsburgh)

  • Isadora Carnaval Detweiler

    (University of Pittsburgh)

  • Jochen Kuper

    (University of Würzburg)

  • Florian Sauer

    (University of Würzburg)

  • Nina Simon

    (Ludwig Maximillian University of Munich)

  • Johanna Bretzler

    (Ludwig Maximillian University of Munich)

  • Simon C. Watkins

    (University of Pittsburgh)

  • Thomas Carell

    (Ludwig Maximillian University of Munich)

  • Caroline Kisker

    (University of Würzburg)

  • Bennett Van Houten

    (UPMC Hillman Cancer Center
    University of Pittsburgh)

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.

Suggested Citation

  • Emily C. Beckwitt & Sunbok Jang & Isadora Carnaval Detweiler & Jochen Kuper & Florian Sauer & Nina Simon & Johanna Bretzler & Simon C. Watkins & Thomas Carell & Caroline Kisker & Bennett Van Houten, 2020. "Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-15168-1
    DOI: 10.1038/s41467-020-15168-1
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