Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome

Abstract

DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.

Fig. 1.

Ribbon model of complex A from A. t. cry3 and the repaired CPD damage in sticks representation with SIGMAA-weighted F_obs − F_calc omit electron density (light blue, contoured at 2 σ) defining the oligonucleotide. The catalytic domain (dark red) of A. t. cry3 contains the catalytic FAD cofactor (yellow) and the antenna chromophore MTHF (orange) in the contact region to the antenna domain (blue). Nomenclature and definition of secondary structure elements are given in Brudler et al. (12).

Fig. 2.

Interactions between A. t. cry3 and the DNA backbone. (A) Structural movements in the active site upon substrate binding to A. t. cry3. The loop region α15–α16 moves up to 4.6 Å and allows several stabilizing interactions between the DNA and the side chains of E444 and R446 as well as between the main chain of D445 and the 2 thymines T1 and T4. The ribbon models show A. t. cry3 with bound DNA (red) and the free protein (pdb code: 2J4D; green). The T5 oligonucleotide (yellow) is shown in stick presentation together with its molecular surface. The formacetal linkage within the synthetic CPD lesion that was split into 2 thymine residues by X-ray radiation is marked as P⁰. Notably, the side chain of R446 is close enough to form a salt bridge with the intralesion phosphate moiety. (B) Comparison of active sites between cry3 (purple) and the class I photolyase from A. n. (cream) complexed to CPD lesion comprising DNA (PDB code 1TEZ). Note that K452 in A. t. cry3 and K414 in A. n. photolyase are noncorresponding residues but occupy very close spaces.

Fig. 3.

Isoelectric surface potential of A. t. cry3 bound to the single-stranded pentameric DNA containing a CPD analog. (A) Top and side (Inset) views. (B and C) Hydration and electrostatics of the active site in the substrate-bound state of cry3. The black arrows in C indicate the water molecules intruded into the active site because of replacement of a tryptophan conserved in class I CPD photolyases by Y434.

Fig. 4.

Binding of cry3 to DNA probes containing a single T<>T dimer in the central position. (A) Sequences and structures of probes. The T<>T dimer is positioned within the VspI recognition site (boxed in probe 1). Probe 1 forms a perfect duplex. In probes 2 and 3, the 5′ and 3′ thymines, respectively, of the T<>T dimer are not hydrogen bonded to the complementary strand. In probe 3, only one hydrogen bond is formed between the 5′ thymine of the T<>T dimer and the complementary adenine (23). In probes 4–8, the T<>T lesion is positioned in the center of loop structures with 2–10 base pairs. Hydrogen bonds between complementary bases are shown as dashed lines. The upper strand (50 nt) was labeled at the 5′ position with IRDye700 (MWG Biotech AG) (marked with asterisk). (B and C) EMSA showing cry3 binding to probes with (B) or without (C) the central T<>T dimer. Probes shown in A and the single-stranded control (probe 9) were mixed with cry3 (+) or with the same aliquot of buffer (−). Arrows indicate the positions of shifted bands. Representative gels from 2 independent experiments are shown. (D) Quantitative binding data. Mean values and standard errors of the 2 independent experiments are shown.

Fig. 5.

Repair of T<>T dimers by cry3. (A) Single-stranded oligo(dT)₁₈. The reaction mixture contained 8.9 μM T<>T dimers in 10 μM oligo(dT)₁₈ and either 10 nM cry3 (squares) or the same aliquot of buffer (circles). Mixtures were treated with photoreactivating light (open symbols) or kept in the dark (filled symbols) at 10 °C. Data represent the mean value of 2 independent experiments, with error bars indicating standard errors. The rate of cry3-catalyzed photorepair was calculated by using the slope of its linear part between 20 and 60 min (solid line) obtained by the least-square method. (B) Quantitative data of repair of a single T<>T dimer in loop structure probes. Data for probes 1–6 (shown in Fig. 4A) are based on the gels shown in Fig. S3.