A baculovirus photolyase with DNA repair activity and circadian clock regulatory function.

Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses.

The Potorous CPD photolyase rescues a cryptochrome-deficient mammalian circadian clock.

Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9) s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures.

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV.

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH(-)) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH(-)). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: toward a detailed understanding of the triple tryptophan electron transfer chain.

The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the approximately 15 A distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH(o)), electron transfer through the chain leads to formation of fully reduced flavin (FADH(-); required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306(o)(+) is reduced either by an extrinsic reductant or by reverse electron transfer from FADH(-). Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH(-) W306(o)(+) was found at 19 +/- 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)(3)](2+). With this yield, the optical spectrum of the excited state of FADH(o) can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain.

What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions.

Cryptochromes and DNA photolyases are highly homologous flavoproteins that accomplish completely different tasks. While plant cryptochrome1 functions as blue light photoreceptor that triggers various morphogenic reactions, photolyases repair UV-induced DNA damages. Both enzymes share the photoactive cofactor, noncovalently bound FAD. For photolyase, the reaction mechanism involves electron transfer to the substrate from the excited-state of fully reduced flavin. For cryptochrome, photoexcitation of the oxidized flavin leads to formation of the semireduced radical FADH(*). Key parameters for the redox state of the flavin in the cell are the midpoint potentials E(1) and E(2) for the oxidized/semireduced and semireduced/fully reduced transitions, respectively. A link between cryptochrome function and its cofactor's redox states has been suggested early on, but no reliable determinations of midpoint potentials have been available. Here we report spectroelectrochemical titrations of cryptochrome1 from Arabidopsis thaliana and photolyases from both E. coli and Anacystis nidulans at pH 7.4. For the cryptochrome, we obtained E(1) approximately E(2) approximately -160 mV vs NHE, strongly deviating from the photolyases where FADH(*) could not be oxidized up to 400 mV, and E(2) approximately -40 mV. Functional and evolutionary implications are discussed, highlighting the role of an asparagine-to-aspartate replacement close to N5 of the flavin.

Electron hopping through the 15 A triple tryptophan molecular wire in DNA photolyase occurs within 30 ps.

DNA photolyase is a photoactive flavoprotein that contains three tryptophan residues between the FAD cofactor and the protein surface, the solvent-exposed Trp being located 14.8 A from the flavin. Photoreduction of the neutral radical FADH. form to the catalytically active FADH- form occurs via electron transfer through this chain. The first step in this chain takes 30 ps, the second less than 4 ps. Using a combination of site-directed mutagenesis and femtosecond polarization spectroscopy to discriminate the spectroscopically indistinguishable Trp residues, we show that the third step occurs in less than 30 ps. This implies that the first photoreduction step is rate limiting and that the Trp chain effectively acts as molecular "wire" ensuring rapid and directed long-range charge translocation across the protein. This finding is important for the functioning of the large class of cryptochrome blue-light receptors, where the Trp chain is conserved. In DNA photolyase we make use of the natural photoactivation of the process, but more generally chains of aromatic amino acids may allow very fast long-range electron transfer also in nonphotoactive proteins.

Active DNA photolyase encoded by a baculovirus from the insect Chrysodeixis chalcites.

The genome of Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) contains two open reading frames, Cc-phr1 and Cc-phr2, which encode putative class II CPD-DNA photolyases. CPD-photolyases repair UV-induced pyrimidine cyclobutane dimers using visible light as an energy source. Expression of Cc-phr2 provided photolyase deficient Escherichia coli cells with photoreactivating activity indicating that Cc-phr2 encodes an active photolyase. In contrast, Cc-phr1 did not rescue the photolyase deficiency. Cc-phr2 was overexpressed in E. coli and the resulting photolyase was purified till apparent homogeneity. Spectral measurements indicated the presence of FAD, but a second chromophore appeared to be absent. Recombinant Cc-phr2 photolyase was found to bind specifically F0 (8-hydroxy-7,8-didemethyl-5-deazariboflavin), which is an antenna chromophore present in various photolyases.. After reconstitution, FAD and F0 were present in approximately equimolar amounts. In reconstituted photolyase the F0 chromophore is functionally active as judged from the increase in the in vitro repair activity. This study demonstrates for the first time that a functional photolyase is encoded by an insect virus, which may have implications for the design of a new generation of baculoviruses with improved performance in insect pest control.

Polarized transient absorption to resolve electron transfer between tryptophans in DNA photolyase.

Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].

50 years thymine dimer.

Fifty years ago thymine dimer was discovered in the Biochemical and Biophysical Laboratory of Delft Technological University, The Netherlands, by one of the authors of this review (Beukers) as the first environmentally induced DNA lesion. It is one of the photoproducts formed between adjacent pyrimidine bases in DNA by UV irradiation, currently known as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Major lesions found in DNA after in vitro or in vivo UV irradiation are the cis-syn cyclobutane thymine dimer and the thymine-cytosine (6-4) photoproduct. Even after 50 years the study of photo-induced DNA lesions is still going on as is illustrated by the hundreds of papers published every year and the millions hits when browsing the internet for dimer-related information. Living organisms possess efficient and different mechanisms to repair detrimental lesions in their DNA. A unique mechanism to repair CPDs is reversion by either direct interaction with light of short wavelength or by enzymatic photoreactivation. Photophysical mechanisms that induce and reverse molecular bonds in biological macromolecules have been a main focus of research of the group in Delft in the middle of the last century. This review describes the break-through results of these studies which were the result of intense interactions between scientists in the fields of physics, organic chemistry and biochemistry. Philosophically, the "view" of the group in Delft was very appealing: since in nature photolesions are induced in DNA by the sun, how is it possible that repair of these lesions could be accomplished by the same energy source. Evolutionary, it is hardly possible to think of a more efficient repair mechanism.

Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain.

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.

Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant.

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.

Powerful skin cancer protection by a CPD-photolyase transgene.

BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.

Crystal structure of a photolyase bound to a CPD-like DNA lesion after in situ repair.

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.

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells.

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.

Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an "inverse" deuterium isotope effect.

We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH(-) and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (k(H)/k(D)=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH(-) (electron transfer time constant tau(et) in the order of 10-50 micros), and one where electron transfer from FADH(-) (tau(et)=25 ms) is followed by reprotonation of the W306 anion.

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation.

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH*) induces an electron transfer over approximately 15 A from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* <-- W382 <-- W359 <-- W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant tau approximately 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (tau approximately 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield ( approximately 0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (tau approximately 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice.

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous beta-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions.