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Tethering-facilitated DNA 'opening' and complementary roles of ?-hairpin motifs in the Rad4/XPC DNA damage sensor protein.


ABSTRACT: XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively 'opening' these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a 'kinetic gating' mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how 'opening' is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed 'open' undamaged DNA in solution and that such 'opening' can largely occur without one or the other of the ?-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound 'open' DNA adopts multiple conformations in solution notwithstanding the DNA's original structure or the ?-hairpins. Molecular dynamics simulations reveal compensatory roles of the ?-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA 'opening' of undamaged sites and the dynamic nature of 'open' DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

SUBMITTER: Paul D 

PROVIDER: S-EPMC7708039 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

Paul Debamita D   Mu Hong H   Tavakoli Amirrasoul A   Dai Qing Q   Chen Xuejing X   Chakraborty Sagnik S   He Chuan C   Ansari Anjum A   Broyde Suse S   Min Jung-Hyun JH  

Nucleic acids research 20201201 21


XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively 'opening' these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a 'kinetic gating' mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how '  ...[more]

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