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  • Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein
Title: Tethering-facilitated DNA ‘opening’ and complementary roles of β-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 β-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 β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-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.  more » « less
Award ID(s):
1715649 2107527
NSF-PAR ID:
10294029
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
48
Issue:
21
ISSN:
0305-1048
Page Range / eLocation ID:
12348 to 12364
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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