DNA curtain assay

For the DNA curtain assay, the total internal reflection fluorescence microscope combined with a fluidic system was built-up as described previously (25). A nano-patterned fused silica slide was assembled into a flowcell containing microchamber. Liposomes consisting of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 0.5% biotinylated-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)) and 8% mPEG 2000-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) (Avanti Polar Lipids) were deposited to the surface of flowcell to form a lipid bilayer (25). A total of 0.1 mg/ml of anti-digoxigenin (11214667001, Roche) was injected and adsorbed on the pentagonal nano-structure. After further surface passivation by 0.4% of bovine serum albumin, 1 mg/ml streptavidin was introduced. Then either undamaged or CPD-containing λ-DNA molecules tagged with biotin and digoxigenin at opposite ends were anchored on individual lipid molecules via biotin-streptavidin linkage. Under the continuous flow, the digoxigenin at the other end was tethered to the pentagonal nano-barrier. Thereby the DNA molecules remained stretched when the flow was turned off (Figure ​(Figure1A1A).

Schematic of DNA curtain assay with quantum dot (Qdot)-conjugated XPC and initial binding position on undamaged lambda (λ) DNA. (A) Schematic of DNA curtains. Top left: top view of DNA curtains, bottom left: side-view of DNA curtains, and right: Qdot-conjugated XPC. The structure of XPC is adopted from yeast Rad4-Rad23 (10). (B) Histogram for the initial binding positions of XPC-RAD23B on undamaged λ-DNA. The error bars were obtained by bootstrapping with 70% confidence interval.

All XPC-RAD23B experiments on the DNA curtain were carried out in the XPC buffer (25 mM Tris–HCl [7.5] with 40 mM, 100 mM or 150 mM NaCl) at 23°C. XPC-RAD23B was fluorescently labeled by FLAG-antibody-conjugated quantum dot (Qdot), which was prepared from a commercial kit (S10454, Thermo Fisher Scientific). XPC-RAD23B and FLAG-antibody-Qdots were mixed at 1 to 40 molar ratio and then incubated on ice for at least 15 min. Excessive Qdots ensured that only one XPC-RAD23B is conjugated with a single Qdot. About 0.5–1 nM XPC-RAD23B was injected into the flowcell. For the collision experiments, 3×FLAG-tagged EcoRI^E111Q and FLAG-antibody-Qdot having different emission wavelength (605 nm) were conjugated at 1–10 molar ratio on ice for at least 15 min. Then 3 nM EcoRI^E111Q was injected into the flowcell and incubated with DNA in EcoRI buffer (40 mM Tris–HCl [7.5], 50 mM NaCl and 2 mM MgCl₂). After unbound EcoRI^E111Q was completely washed out, 1 nM XPC-RAD23B tagged with Qdot (705 nm emission) was injected in the XPC buffer containing 150 mM NaCl. When the maximum amount of proteins reached the DNA curtains, the flow was turned off and data were collected through NIS-elements (Nikon) with 100 ms exposure time for 5 min. Although all Qdots were conjugated with FLAG antibodies and both proteins had FLAG tags, we did not observe any adhesion of two Qdots on DNA, ensuring that there was no serious cross binding between proteins and Qdots.

For the binding test of XPC-RAD23B on the CPD-containing λ-DNA, single-tethered DNA curtain assay was performed, in which the biotinylated end of λ-DNA is anchored on lipid bilayer and the opposite end is free. To stretch the CPD-containing λ-DNA, XPC buffer with 150 mM NaCl was continuously flowed in. A total of 1 nM Qdot-conjugated XPC-RAD23B was injected. After 10 min incubation with the damaged λ-DNA in the absence of flow, the binding of XPC-RAD23B was imaged under the flow.