Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.     

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.     

The second chromophore in Drosophila photolyase/cryptochrome family photoreceptors

The photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light-dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photo-antenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date, no second chromophore has been identified in cryptochromes. Drosophila contains three members of the cryptochrome/photolyase family: cyclobutane pyrimidine dimer (CPD) photolyase, (6-4) photoproduct photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/photolyase family members. Using this system, we demonstrate that Drosophila CPD photolyase and (6-4) photolyase employ 5-deazariboflavin as their second chromophore, but Drosophila cryptochrome, which is evolutionarily closer to (6-4) photolyase than the CPD photolyase, lacks a second chromophore.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.     

Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis

Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.     

Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6-4) photolyase as revealed by resonance Raman spectroscopy: Electron transfer from the FAD cofactor to ultraviolet-damaged DNA

The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm(-1) bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm(-1) band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N5-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N3-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997     

NMR study of repair mechanism of DNA photolyase by FAD-induced paramagnetic relaxation enhancement

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.     

The native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.     

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.     

DNA photorepair: chromophore composition and function in two classes of DNA photolyases

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.     

Photoreactivation of (6-4) photolyase in Dunaliella salina.

Dunaliella salina is a unicellular green alga and possesses two types of photolyase: Class II cyclobutane pyrimidine dimers (CPD) photolyase and (6-4) photolyase. The gene of D. salina (6-4) photolyase is the first one found in unicellular organisms. CPD photolyases have been extensively studied but (6-4) photolyases are less understood. Because of the data observed in this study, D. salina (6-4) photolyase is insensitive to high salinity; whether it can tolerate a higher level of salinity than other (6-4) photolyases needs to be studied further. However, evidence is provided that (6-4) photolyases might be highly conserved among different species, not only in the sequence identity but also in the photorepair mechanism.     

Identification of a Novel Class of Photolyases as Possible Ancestors of Their Family

UV irradiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These two types of lesions can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. Recently, a new class of 6-4 photolyases named iron–sulfur bacterial cryptochromes and photolyases (FeS-BCPs) were found, which were considered as the ancestors of all photolyases and their homologs—cryptochromes. However, a controversy exists regarding 6-4 photoproducts only constituting ∼10–30% of the total UV-induced lesions that primordial organisms would hardly survive without a CPD repair enzyme. By extensive phylogenetic analyses, we identified a novel class of proteins, all from eubacteria. They have relatively high similarity to class I/III CPD photolyases, especially in the putative substrate-binding and FAD-binding regions. However, these proteins are shorter, and they lack the “N-terminal α/β domain” of normal photolyases. Therefore, we named them short photolyase-like. Nevertheless, similar to FeS-BCPs, some of short photolyase-likes also contain four conserved cysteines, which may also coordinate an iron–sulfur cluster as FeS-BCPs. A member from Rhodococcus fascians was cloned and expressed. It was demonstrated that the protein contains a FAD cofactor and an iron–sulfur cluster, and has CPD repair activity. It was speculated that this novel class of photolyases may be the real ancestors of the cryptochrome/photolyase family.     

PHL1 of Cercospora zeae-maydis encodes a member of the photolyase/cryptochrome family involved in UV protection and fungal development

DNA photolyases harvest light energy to repair genomic lesions induced by UV irradiation, whereas cryptochromes, presumptive descendants of 6-4 DNA photolyases, have evolved in plants and animals as blue-light photoreceptors that function exclusively in signal transduction. Orthologs of 6-4 photolyases are predicted to exist in the genomes of some filamentous fungi, but their function is unknown. In this study, we identified two putative photolyase-encoding genes in the maize foliar pathogen Cercospora zeae-maydis: CPD1, an ortholog of cyclobutane pyrimidine dimer (CPD) photolyases described in other filamentous fungi, and PHL1, a cryptochrome/6-4 photolyase-like gene. Strains disrupted in PHL1 (Deltaphl1) displayed abnormalities in development and secondary metabolism but were unaffected in their ability to infect maize leaves. After exposure to lethal doses of UV light, conidia of Deltaphl1 strains were abolished in photoreactivation and displayed reduced expression of CPD1, as well as RAD2 and RVB2, orthologs of genes involved in nucleotide excision and chromatin remodeling during DNA damage repair. This study presents the first characterization of a 6-4 photolyase ortholog in a filamentous fungus and provides evidence that PHL1 regulates responses to UV irradiation.     

Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA

Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA. We solved crystal structures of Mm0852, the first one for a class II photolyase, alone and in complex with CPD lesion-containing duplex DNA. The lesion-binding mode differs from other photolyases by a larger DNA-binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophane dyad as electron transfer pathway to the catalytic FAD cofactor.     

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.     

Influence of a cis,syn‐cyclobutane pyrimidine dimer damage on DNA conformation studied by molecular dynamics simulations

The photo‐induced formation of cis‐syn‐cyclobutane pyrimidine dimers (CPD) is a highly mutagenic and cancerogenic DNA lesion. In bacteria photolyases can efficiently reverse the dimer formation employing a light‐driven reaction after looping out the CPD damaged bases into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The CPD damage may alter the DNA structure and dynamics already in the absence of the repair enzyme which can facilitate the initial binding of a photolyase repair enzyme. To characterize the effect of a CPD damage, extensive comparative molecular dynamics (MD) simulations on duplex DNA with central regular or CPD damaged nucleotides were performed supplemented with simulations of the DNA‐photolyase complex. Although no spontaneous flipping out transitions of the damaged bases were observed, the simulations showed significant differences in the conformational states of regular and CPD damage DNA. The isolated damaged DNA adopted transient conformations which resembled the global shape of the repair enzyme bound conformation more closely compared to regular B‐DNA. In particular, these conformational changes were observed in most of helical and structural parameters where the protein bound DNA differs drastically from regular B‐DNA. It is likely that the transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out process of the damaged DNA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 215–222, 2015.     

Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor.

The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.     

Computational analysis of DNA photolyases using digital signal processing methods

Sun light energy is used by plants to trigger their growth and development. However, an increase of UV-B light may lead to DNA damage. DNA photolyases are enzymes that repair the cyclobutane pyridine dimer (CPD) and 6–4 photoproduct lesions formed through UV irradiation of DNA. Many aspects of the repair process are under intense scientific investigation but still poorly understood. Here we have computationally analysed DNA-photolyases using the resonant recognition model (RRM), a physico-mathematical approach based on digital signal processing methods. The RRM proposes that protein interactions represent the transfer of resonant electromagnetic energy between interacting molecules at the particular frequency. Within this study we have determined photolyases characteristic frequency, “hot spots” amino acids corresponding to the functional mutations and functional active/binding sites, and designed photolyase peptide analogous. A mutual relationship between photolyase and p53 tumour suppressor protein has also been investigated. The results obtained provide new insights into the structure–function relationships of photolyase protein family.