Search for: All records

Abstract Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. Using laser temperature-jump (T-jump) spectroscopy combined with cytosine-analog Förster Resonance Energy Transfer (FRET) probes that sense local DNA conformations, we measured the intrinsic dynamics of DNA containing 3 base-pair mismatches recognized in vitro by Rad4 (yeast ortholog of XPC). Rad4/XPC recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. T-jump measurements, together with a novel and rigorous comparison with equilibrium FRET, uncovered conformational dynamics spanning multiple timescales and revealed key differences between Rad4-specific and non-specific DNA. AT-rich non-specific sites (matched or mismatched) exhibited dynamics primarily within the T-jump observation window, albeit with some amplitude in ‘missing’ fast (<20 μs) kinetics. These fast-kinetics amplitudes were dramatically larger for specific sites (CCC/CCC and TTT/TTT), which also exhibited ‘missing’ slow (>50 ms) kinetics at elevated temperatures, unseen in non-specific sites. We posit that the rapid (μs–ms) intrinsic DNA fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-ms kinetics in specific DNA reflect a propensity to adopt unwound/bent conformations resembling Rad4-bound DNA structures. These studies provide compelling evidence for sequence/structure-dependent intrinsic DNA dynamics and deformability that likely govern damage sensing by Rad4. 

Xeroderma pigmentosum C (XPC) is a key initiator in the global genome nucleotide excision repair pathway in mammalian cells. Inherited mutations in the XPC gene can cause xeroderma pigmentosum (XP) cancer predisposition syndrome that dramatically increases the susceptibility to sunlight-induced cancers. Various genetic variants and mutations of the protein have been reported in cancer databases and literature. The current lack of a high-resolution 3-D structure of human XPC makes it difficult to assess the structural impact of the mutations/genetic variations. Using the available high-resolution crystal structure of its yeast ortholog, Rad4, we built a homology model of human XPC protein and compared it with a model generated by AlphaFold. The two models are largely consistent with each other in the structured domains. We have also assessed the degree of conservation for each residue using 966 sequences of XPC orthologs. Our structure- and sequence conservation-based assessments largely agree with the variant’s impact on the protein’s structural stability, computed by FoldX and SDM. Known XP missense mutations such as Y585C, W690S, and C771Y are consistently predicted to destabilize the protein’s structure. Our analyses also reveal several highly conserved hydrophobic regions that are surface-exposed, which may indicate novel intermolecular interfaces that are yet to be characterized. 

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities. Here we report that photocleavable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically recognizes diverse helix-destabilizing/distorting lesions including bulky organic adduct lesions and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding causes it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair. 

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.