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bioRxiv · 10.1101/2020.09.28.313049

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

Paul, D.·Mu, H.·Tavakoli, A.·Dai, Q.·Chen, X.·Chakraborty, S.·He, C.·Ansari, A.·Broyde, S.·Min, J. H.
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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 {beta}-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound open DNA adopts multiple conformations in solution notwithstanding the DNAs original structure or the {beta}-hairpins. Molecular dynamics simulations reveal compensatory roles of the {beta}-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 NER in cells.

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Paul, D., Mu, H., Tavakoli, A., Dai, Q., Chen, X., Chakraborty, S., He, C., Ansari, A., Broyde, S., Min, J. H.. 2020-09-29. Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein. https://doi.org/10.1101/2020.09.28.313049

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