Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

Abstract

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

“…Furthermore, the amplitude of the rapid (unresolved) kinetics was diminished in the complex (SI Appendix, Fig. S10), again reminiscent of our previous observations with 2AP constructs (49), indicating that mismatch premelting is partially suppressed in the presence of Rad4. The relaxation kinetics in the T-jump time window are detected only in the presence of the protein and require both the donor and the acceptor in AN12; they were not detected in the absence of the protein (Fig.…”

“…The rapid increase in AN12_DA is in a direction opposite of the rapid decrease seen in AN12_D; instead, it mirrors the increase in I DA seen in equilibrium measurements on the AN12_DA sample (Fig. 3B, pink) and is reminiscent of the rapid kinetics of mismatch unstacking ("premelting") previously observed using 2AP-labeled DNA (49). We thus propose that the rapid increase in AN12's I DA after the T-jump reflects similar premelting kinetics, as sensed by the rapid reorientation of tC nitro adjoining the mismatch bubble.…”

“…Structure. We have previously reported T-jump measurements of Rad4-induced DNA opening kinetics, probed specifically at mismatch model lesion sites using fluorescent 2-aminopurine (2AP) as one of the mismatched nucleotides (49). Here, we aimed at probing motions that affect regions beyond the mismatched nucleotides such as local unwinding around the lesion, as observed in the open structures (Figs.…”

“…These lesions are structurally diverse, but the damage is confined on one strand of the duplex DNA and commonly accompanies local helix destabilization/distortion. In vitro, Rad4/XPC can also specifically bind to 2-bp or 3-bp mismatch DNA (49,53). The crystal structures of Rad4 bound to UV-induced model lesions showed that Rad4 inserts a β-hairpin into the lesion site and flips out two damage-containing nucleotide pairs; the duplex around the lesion is also partially unwound and bent (54) (Fig.…”

Architecture of the human XPC DNA repair and stem cell coactivator complex

“…This similarity between different DNA-bound structures is also consistent with recent work describing a kinetic but not structural means of discrimination between damaged and undamaged DNA by Rad4 (41). The reduction of density in our DNA-bound structures compared with the apo XPC complex reflects a conformational change consistent with two phenomena observed in the Rad4 crystal structure: (i) the C-terminal portion of Rad4 shifting toward the DNA substrate, and in the context of our docking, away from the region of reduced density (Fig.…”