Validation of XP-C pathogenic variations in archival material from a live XP patient

McDaniel, Lisa D.; Rivera-Begeman, Amanda; Doughty, Ana T.B.; Schultz, Roger A.; Friedberg, Errol C.

doi:10.1016/j.dnarep.2006.09.009

“…Mutational analysis were performed by genomic DNA sequencing using primer pairs flanking the intron exon boundaries to ensure that disease-causing mutations are detected within the exon and the intron regions. In the XPC gene 46 different disease-causing mutations, distributed over the whole gene (16 exons), have been identified so far (McDaniel et al, 2007).…”

Section: Mutational Analysis Of Xp-c Fibroblastsmentioning

confidence: 99%

Clinical, functional, and genetic analysis of NER defective patients and characterization of five novel XPG mutations

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

Paul

¹

,

Mu

²

,

Tavakoli

³

et al. 2020

Nucleic Acids Research

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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.

show abstract

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

Paul

¹

,

Mu

²

,

Tavakoli

³

et al. 2020

Preprint

View full text Add to dashboard Cite

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 NER in cells.

show abstract

Validation of XP-C pathogenic variations in archival material from a live XP patient

Cited by 4 publications

References 17 publications

Clinical, functional, and genetic analysis of NER defective patients and characterization of five novel XPG mutations

Clinical, functional, and genetic analysis of NER defective patients and characterization of five novel XPG mutations

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

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

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