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.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

“…These characteristics of AT10_DA agree well with those of other matched DNA duplexes we had previously examined and confirm that AT10 mainly adopts B-DNA conformation with perhaps a minor population of non-B-DNA conformations. 43,50 Also, Rad4-binding to the DNA did not alter the FLT profile, as shown previously with other nonspecific DNA, indicating that nonspecific binding by Rad4 does not lead to detectable changes in tC1-tC nitro -based FRET (Fig. 4A and Fig.…”

“…3). 40,41,43,47,50,51 In this assay, the binding of the protein to 5 nM 32 40 On the other hand, the NPOM-DNA after photocleavage (AT2 + hn) showed K d,app (B744 nM) comparable to that of the unmodified DNA (AT1). These results show that the NPOM adduct in DNA is specifically recognized by Rad4/XPC as a lesion and that its photoremoval abolishes the specific binding.…”

“…43 Furthermore, its specific binding to Rad4 further decreased the average FRET, a direction of change in line with expected FRET changes based on the known DNA conformation in the 'open' crystal structure. 43,50 These results indicate an intriguing possibility that the conformation of NPOM-DNA when specifically bound to Rad4 may be different from those of CCC/CCC or other DNA lesions that form the 'open' conformation.…”

“…S7A, ESI †). 43,50,67 In comparison, the DNA containing both the donor and acceptor (AT10_DA) showed a major lifetime peak (t DA ) at 0.27 ns with 86% fractional population with minor peaks at 1.8 ns (7%) and 4.8 ns (7%) (Fig. 4A and Fig.…”

“…To determine the relative affinities of Rad4 binding to different DNA substrates, competition EMSA (or gel-shift assays) were employed as previously described. 40,41,43,47,50,51 The benefit of using this competition assay over the conventional singlesubstrate EMSA is that one can directly observe any preferential binding over the nonspecific binding, including factors such as potential DNA end-binding while avoiding multiple proteins aggregating on a single DNA, as is the case when protein is in excess of total DNA. 51 Various concentrations of the Rad4-Rad23 complexes were mixed with 5 nM 32 P-labelled DNA of interest (mismatched/damaged or matched/undamaged) in the presence of 1000 nM cold (unlabeled), matched DNA, CH7_NX in an EMSA buffer (5 mM BTP-HCl, 75 mM NaCl, 5 mM DTT, 5% glycerol, 0.74 mM 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), 500 mg ml À1 bovine serum albumin, pH 6.8).…”