DNA photolyases use light energy to repair DNA that comprises ultraviolet-induced lesions such as the cis-syn cyclobutane pyrimidine dimers (CPDs). Here we report the crystal structure of a DNA photolyase bound to duplex DNA that is bent by 50 degrees and comprises a synthetic CPD lesion. This CPD lesion is flipped into the active site and split there into two thymines by synchrotron radiation at 100 K. Although photolyases catalyze blue light-driven CPD cleavage only above 200 K, this structure apparently mimics a structural substate during light-driven DNA repair in which back-flipping of the thymines into duplex DNA has not yet taken place.

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-siturepaired 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.Arabidopsis ͉ DNA repair ͉ photolyase C ryptochromes (cry) and DNA photolyases form a unique family of flavoproteins, with members present in all kingdoms of life (1). This family is divided into several subclades according to sequence similarity and function. DNA photolyases are enzymes that repair cytotoxic and mutagenic DNA lesions induced by UV-B, specifically cis-syn cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone lesions (6-4 photoproduct) by using light energy in the UV-A/blue region (2). The catalytic cofactor of DNA photolyase is flavin adenine dinucleotide (FAD) in its fully reduced form (FADH Ϫ ), and this is present in a U-shaped conformation, as shown in several DNA photolyase structures (3-7). Catalysis involves electron transfer from the excited catalytic cofactor to the UV-B photoproduct, splitting the cyclobutane or oxetane rings, and electron backtransfer to the semireduced FADH°(1). Excitation of FAD is accomplished either by direct photon absorption or by Förster-type energy transfer from an antenna cofactor (1). Despite considerable sequence and structural similarity with DNA photolyases and common cofactor compositions, cryptochromes generally lost repair activity but gained photoreceptor function operating in the same waveband region as DNA photolyases (1). In plants, cryptochromes trigger several developmental processes, such as deetiolation and photoperiodic flower induction, and they entrain the circadian clock (8). In animals such as Drosophila, cryptochromes also function as photoreceptors for light input to the clock (9) and in magnetoreception (10), whereas mammalian cryptochromes are central components of the circadian clock without proven photoreceptor function (11). The more recently discovered subclade of the cryptochrome/ photolyase family, named cry DASH, includes members in plants, cyanobacteria, eubacteria, and vertebrates (12-15). It has been suggested that they represent photoreceptors because they lacked repair activity for CPDs in double-stranded DNA (dsDNA), despite their DNA-binding activity (12, 13). Positive support fo...

DNA photolyases are highly efficient light-driven DNA repair enzymes which revert the genome-damaging effects caused by ultraviolet (UV) radiation. These enzymes occur in almost all living organisms exposed to sunlight, the only exception being placental mammals like humans and mice. Their catalytic mechanism employs the light-driven injection of an electron onto the DNA lesion to trigger the cleavage of cyclobutane- pyrimidine dimers or 6-4 photoproducts inside duplex DNA. Spectroscopic and structural analysis has recently yielded a concise view of how photolyases recognize these DNA lesions involving two neighboring bases, catalyze the repair reaction within a nanosecond and still achieve quantum efficiencies of close to one. Apart from these mechanistic aspects, the potential of DNA photolyases for the generation of highly UV-resistant organisms, or for skin cancer prevention by ectopical application is increasingly recognized.