Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

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.

The data show that V131C/C132S mutations did not affect DNA binding of Rad4. The ΔBHD3 and Δβ-hairpin3 mutants, however, exhibit significantly weakened binding to mismatch DNA while retaining affinities to undamaged DNA, resulting in significant loss in lesion recognition specificity. a. The crosslinked Rad4-Rad23-DNA complex was purified over a MonoQ column (GE Healthcare) with 0-2 M NaCl gradient. The separated peaks corresponding to the crosslinked complex and the free protein and DNA are indicated with arrows.

Supplementary
b. Non-reducing SDS-PAGE gels show that the crosslinked complex (Lane 5,6,7) were separated from the free proteins (Lane 2, 3). To first quench the crosslinking reaction, all the samples were mixed with 0.1 mM of S-methyl methanethiosulfonate (Sigma) and incubated at room temperature for 10 min. Subsequently the samples were loaded to 15% SDS-PAGE gel using a loading buffer lacking 2-mercaptoethanol. Gels were run at 180 V for 50 min. Lane 1 contains crosslinking reaction mixture before purification; lane 2 and 3 shows free protein band eluting at 280-320 mM NaCl; lane 5-8 shows the crosslinked complex eluting at 400-480 mM NaCl.
c. Crystal photo of the Rad4-Rad23 complex crosslinked to 24-bp undamaged duplex DNA (Fig.  1). The crystal was grown in 5 mM BTP-HCl, 200 mM NaCl, 15.5% isopropanol, 100 mM CaCl 2 , pH 6.8 . b. Histogram of the internal binding position of Rad4 along DNA. The positions on DNA were measured as the shortest distance from protein to DNA end and calculated by percent of total DNA length. A total of 67 molecules were measured. The apparently low frequency of Rad4 binding near the end of DNA (10-20% position) is due to the exclusion of molecules with protein bound at an end as well as to the resolution limit that hampers the detection of very short segments of DNA extending beyond a bound protein. Aside from the end-region of the DNA, however, the distribution of Rad4 is mostly random throughout the rest of the DNA segment (20-50% position). The only exception seems to be at position ~ 47%, which appears to have less Rad4 bound than at other internal regions. There could be multiple reasons why internally bound Rad4 may exhibit certain degree of non-randomness. For instance, it may be due to sequence-dependent preference of Rad4 for certain stretches of DNA versus others. Such preference may arise from any propensity of the DNA sequence to be more easily bent, unwound, and/or opened up by Rad4 to form the "open" conformation. 2AP fluorescence measurements similar to those reported for AN3 (Fig. 4) were carried out for another mismatched sequence, AN21 (Supplementary Table 1). AN21 contains a 2-bp mismatch and more closely resembles the sequence used in crystallographic studies.
a. The 2AP fluorescence emission spectra measured for DNA alone (black) and Rad4-DNA complex (red), with excitation at 314 nm at 25 °C. The fluorescence emission intensities for AN21 increased ~10-fold upon Rad4 binding.
b. The maxima of the 2AP fluorescence emission spectra, measured at 365 nm as a function of temperature, for DNA alone (black) and Rad4-DNA complex (red); the intensities are normalized to match at the lowest temperature. Open and filled symbols are for two independent sets of measurements on each sample. Notably, 2AP fluorescence in AN21 alone increases with increasing temperature, in contrast to the behavior observed in AN3, which showed a decrease in 2AP fluorescence with increasing temperature (Fig. 4b, left). Thus, in the AN21 context, 2AP in free DNA has an increased propensity to unstack as the temperature is raised.
c. Relaxation traces measured on the Rad4-DNA complex, in response to a 4 ºC T-jump, show single-exponential kinetics, with relaxation time 8.0 ± 1.9 ms (at final temperature 21 ºC). The relaxation rates measured on the Rad4-AN21 and Rad4-AN3 complexes overlap (Fig. 6), indicating that the rate-limiting step in forming the "open" conformation is not sequence-context dependent.
d. Relaxation kinetics measured on the AN21 DNA-only sample exhibited much slower kinetics, with relaxation time 169 ± 98 ms, similar to T-jump recovery kinetics back to the initial temperature. The uncertainty is the sample standard deviation obtained from two independent measurements. Note that the intensity immediately after the T-jump increases for this sample, consistent with its equilibrium temperature-dependence (panel b), indicating that 2AP unstacking is too fast to be resolved on the ~5 s time-resolution of our T-jump instrument. In contrast, for the Rad4-AN21 complex, the intensity immediately after T-jump drops (panel c), indicating that rapid 2AP unstacking is suppressed in the complex.