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blue light receptor family
Mutation Research 434 Ž1999. 89–97 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres
Minireview
Functional diversity of the DNA photolyaserblue light receptor family Takeshi Todo
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Radiation Biology Center, Kyoto UniÕersity, Yoshida-konoe-cho, sakyoku, Kyoto 606-8501, Japan Accepted 16 March 1999
Keywords: DNA photolyase; CRY; DNA repair, UV damage; Circadian rhythm; Blue light photoreceptor; Cryptochrome
1. Introduction Proteins of the DNA photolyaserblue light photoreceptor family mediate either DNA repair or perceptionrsignal transduction of external light signals. Currently, this protein family is known to consist of three groups; cyclobutane pyrimidine dimer ŽCPD. photolyase, Ž6-4.photolyase and blue light photoreceptor Žcryptochrome: CRY.. Among them, CPD photolyase is the best characterized and its reaction mechanism has been elucidated in considerable detail. Compared with CPD photolyase, the reaction mechanisms of other two groups have been less well studied. The aim of this minireview is to summarize our current understanding of the characteristics of this protein family, focusing especially on the functional diversity and structural similarity between each group and CPD photolyase. Comparison between the structural and functional properties of these proteins will lead to understanding of each individual protein from a different viewpoint, especially by use of the wealth of the knowledge of the precise features of CPD photolyase.
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2. DNA photolyaser r blue light photoreceptor family 2.1. Functional diÕersity Photoreactivation ŽPR. reverses the effect of ultraviolet ŽUV. light when the organism is either concomitantly or subsequently exposed to blue light w1x. CPD and 6-4 wpyrimidine-2X-onex pyrimidines wŽ64.photoproductsx constitute major sources of base damage following the exposure of living cells to UV radiation w2x. Enzymes that are involved in the PR of UV-damaged DNA are called DNA photolyase w3–5x. DNA photolyase binds to UV-damaged DNA and, upon absorbing a blue light photon Žof wavelength 300 to 500 nm. splits CPD, restoring the bases to their native forms. Thus such photolyases are classified as CPD photolyases. CPD photolyase was first cloned from Escherichia coli in 1978 and subsequently the same gene was cloned from several organisms. CPD photolyases are classified into two classes by their primary structure: Class I and Class II CPD photolyases w6,7x. Class I CPD photolyase genes have been isolated from prokaryotes and eukaryotes Žfungi., but all from unicellular organisms w1,4,7x. Class II CPD photolyase genes have been
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isolated from a wide variety of organisms, including eubacteria, archaebacteria and higher eukaryotes w7– 9x. In the history of photolyase research, two new findings were made in 1993. One was the isolation of plant blue light photoreceptor genes that mediate numerous blue light-dependent responses w10x. The blue light photoreceptor gene was first identified as the causative gene for a mutant defective in a blue light sensing pathway mediating inhibition of hypocotyl elongation in Arabidopsis thaliana. This gene was named CRY 1, after cryptochrome, the name commonly given to plant blue-UVA photoreceptors. One more CRY gene Ž CRY 2 . was identified by similarity of its primarily structure to CRY 1 w11x. Mutation of this gene was shown to be responsible for later flowering than wild-type, thus regulating flowering time in response to light. In addition to the regulation of flowering time, CRY 2 shares considerable functional overlap with CRY 1. In seedling development, CRY 1 mediates high-intensity blue light responses and CRY 2 mediates low-light responses w12x. Furthermore, mutant plants lacking both CRY 1 and CRY 2 are deficient in the phototropic response w13x, although neither single mutant shows any phototropic defect. Thus, in plants, the CRY proteins function as photoreceptors for many blue light dependent responses. CRY gene was also isolated from Sinapis alba w14x. The apoproteins of Arabidopsis CRY 1 and 2 and Sinapis CRY are very similar to CPD photolyase and hence are structurally related to the photolyase, although CRY functions in signal transduction, not in DNA repair w15x.
The second was discovery of a new type of DNA photolyase, Ž6-4.photolyase. The CPD photolyase does not repair Ž6-4.photoproducts w1,3,4x. Because of the structural difference of a Ž6-4.photoproduct from a CPD w16x, it was thought that photoenzymatic reversal of Ž6-4.photoproducts was very unlikely for the following reason. The formation of Ž6-4.photoproduct involves the transfer of the hydroxy Žor amino. group at C-4 of the 3X base of the dinucleotide to the C-5 position of the 5X base concomitant with formation of a sigma bond between the C-6 of the 5X base and the C-4 of the 3X base ŽFig. 1.. Therefore, even if an enzyme breaks the 6-4 C–C bond, the bases would not be restored to their original forms. Thus, it had long been thought that Ž64.photoproducts were repaired exclusively by a light-independent repair mechanism, namely nucleotide excision repair. However, in 1993, it was reported that in Drosophila melanogaster there exists a light-dependent DNA repair activity specific for Ž6-4.photoproducts w17x. Later on, similar activity was further observed in some vertebrates and plants, but so far has not been detected in humans or in bacteria such as E. coli w17,18x. Currently, the cDNAs of Ž6-4.photolyase have been cloned from Drosophila w19x, Xenopus w20x, Arabidopsis w21x and Danio rerio ŽZebra fish. ŽY. Kobayashi, unpublished data.. The genes for the Ž6-4.photolyase apoprotein exhibit a sequence similarity to CPD photolyase, in particular to the Class I CPD photolyase w19x. Two genes having 41–45% sequence identity to Drosophila Ž6-4.photolyase are found in the human genome w19,22,23x. Since the proteins encoded by
Fig. 1. Reaction scheme for Ž6-4.photoproduct formation by UV irradiation. The proposed mechanism of Ž6-4.photolyase by the reverse reaction of formation is also shown by the arrow with dashed line.
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these genes does not show any DNA repair activity w22,24x and their functions have been predicted to be similar to those of plant cryptochrome, the proteins have been designated hCRY1 and hCRY2 w22x. A similar type of gene has also been identified in Drosophila and named as dcry w25–29x. These genes are expressed in a wide variety of tissues including inner tissues where light never reaches w19,22,23x. However, the murine homolog of hCRY, mCRY1 and mCRY2, are strongly expressed in suprachiasmatic nucleus ŽSCN. and the retina, respectively w30x, where the master clock and the photoreceptor for circadian rhythm are predicted to exist, respectively. Furthermore, in the former tissue mCRY1 expression oscillates with circadian rhythm w30x. This is the first finding suggesting the involvement of animal CRY in the circadian photoreceptor. Most organisms contain molecular time-keepers known as circadian clocks, which determine their daily biological rhythms w31,32x. Circadian clocks are set by the light and dark cycles of day and night, and once set keep time even in constant darkness. Exposure to light during the dark period can reset the clock w31,32x. Eyes are thought to be an input machinery to the clock, although eyeless and visually blind mutants of Drosophila exhibit normal light responses in circadian rhythm, suggesting that rhodopsins in eyes are not the circadian photoreceptor molecule w33x. In Drosophila, the light-induced phase shift of adult locomotor activity shows wavelength dependency in that maximal effects are observed with light of 400– 500 nm, while light of 600 nm or greater has no effects w33x. This action spectrum implicates the possible involvement of cryptochrome-like molecules in light response as a circadian photoreceptor, since the photolyasercryptochrome family comprises flavin-containing proteins having a characteristic absorption at 400–500 nm Žthe absorption maximum of an oxidated FAD-containing protein is 450 nm.. This possibility was demonstrated to be the case both in mouse w34x and Drosophila w25–27x. In mice, mutation of the cry gene has similar effects on circadian rhythm. Mice lacking mCRY2 were generated w34x. This knock-out mutant have reduced rate of photoinduction of a circadian reporter gene Ž per 1. in addition to the high amplitude phase shift in response to light pulses and the long free-running period w34x. A Drosophila mutant in the gene encod-
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ing dCRY was identified. In this mutant, circadian oscillation of clock gene Ž per and tim. expression disappeared w25x. Furthermore, the mutant shows no response to brief light pulses on resetting the clock Žphase shift of circadian rhythm. w26x. On the other hand, the phase shifts in transgenic flies over-expressing dCRY respond abnormally to light pulses w27x. More recently, it was reported that CRY 1 mutant of Arabidopsis shows the long free-running period under constant blue light w35x. These results indicate that CRY is a photoreceptor dedicated to the control of circadian rhythms. 2.2. Structural similarity Amino acid sequence alignment revealed that Class II CPD photolyase is distantly related to the other members of this family w7x. While members in each group of the protein family show relatively high mutual homology Ž25–80%., a much lower homology of 10–17% is found between each group except for that between Ž6-4.photolyase and animal CRY Ž40–60% homology.. Thus, this protein family can be classified into four groups from their primary structure: Ž1. Class I CPD photolyase, Ž2. Class II CPD photolyase, Ž3. plant CRY and Ž4. Ž6-4.photolyaseranimal CRY, although functionally they are divided into two groups: DNA repair ŽClass I and II CPD photolyase and Ž6-4.photolyase. and photoreceptor Žplant CRY and animal CRY. ŽFig. 2.. All known CPD photolyases contain two chromophores w3,4x. The first one is a reduced flavin adenine dinucleotide ŽFADHy. and second is either methenyltetrahydrofolate or deazaflavin w1,3,4x. The current consensus is that the second chromophore functions as an antenna which is elevated into an energetically excited state after absorbing blue light. This excited antenna molecule then transfers energy to FADHy, which in turn donates an electron to the CPD substrate held in the enzyme’s active site. This negative charge induces splitting of the cyclobutane ring of CPD. Subsequently, the electron is transferred back to flavin, restoring a CPD into its original state of two neighboring pyrimidines w3,4x. This repair reaction occurs even when the second chromophore is depleted from CPD photolyase w36x. In this case FADHy itself absorbs blue light. Thus, the FAD is necessary and sufficient for catalysis. Apoen-
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Fig. 2. Unrooted phylogenetic tree based on alignment of several members of the DNA photolyaserblue light receptor family. Abbreviations of species are: N. crassa ŽNc., S. cereÕisiae ŽSc., E. coli ŽEc., S. typhimurium ŽSt., A. nidurans ŽAn., H. halobium ŽHh., S. griseus ŽSg., A. thaliana ŽAt., S. alba ŽSa., C. reinhardtii ŽCr., M. domestica ŽMd., C. auratus ŽCa., D. melanogaster ŽDm., M. thermoautotrophicum ŽMt., Zebrafish ŽZ., H. sapiens Žh., X. laeÕis ŽXl..
zyme without FAD had no affinity for CPD but did regain its specific binding to CPD upon binding stoichiometrically to FAD w36x. This recovery of DNA binding activity by apophotolyase reconstituted with FAD indicates that this cofactor is structurally important in forming the DNA binding site. Thus, in addition to its essential role in catalysis, FAD also possesses an important role in the protein structure.
Structural features of FAD binding site of CPD photolyase have been analyzed precisely in E. coli CPD photolyase. The crystal structure of E. coli CPD photolyase was solved at a resolution of 2.3A w37x. The three-dimensional structure of CPD photolyase shows that the protein is folded into five b sheets and 20 a helices and these structural elements combine to form two domains, an N-terminal arb
Fig. 3. Domain structure of DNA photolyaserblue light receptor protein family. The domains of chromophore binding site and C-terminal extension are shown on the top. In the middle schematic diagram, conserved regions are shown by filled boxes.
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domain and a C-terminal helical domain. The flavin cofactor residues lies in the C-terminal helical domain and the folate chromophore is in the N-terminal domain ŽFig. 3.. The helical domain can be grouped into two clusters, Cluster I and Cluster II. Clusters I and II interact with the phosphate oxygens and riboflavin part of the FAD, respectively, indicating that the two helical clusters are kept together by binding with FAD to keep the higher structure of enzyme. All other member of this protein family also contain FAD as cofactor w20,22,24,28,29,38x. The second chromophore has not yet been characterized well, although a pterin is suggested to be a second chromophore in S. alba w15x, A. thaliana w13,15x and human CRY w22x. Regions highly conserved between
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all groups of the family exist at N-terminal and C-terminal half ŽHelical Domain. Žfilled box in Fig. 3.. The FAD binding sites identified in E. coli CPD photolyase exist in the highly conserved C-terminal half Žresidues 204–450.. The amino acid residues identified as FAD binding sites in E. coli CPD photolyase are shown in Fig. 4. The residues contacting with FAD are shown in white ŽTyr 222 , Thr 234 , Arg 236 , Leu237, Ser 238 , Trp 271 , Arg 278 , Trp 338 , Asn341, Asp 372 , Asp 374 and Asn378 .. These residues are conserved well among this protein family, although Tyr 222 is substituted by Phe in Ž6-4.photolyase, Arg 236 is substituted by Val in Ž6-4.photolyase, Trp 271 is substituted by Lys in Ž6-4.photolyase and animal CRY and N 341 is substituted by His in Ž6-4.photolyase and animal CRY. Conservation of
Fig. 4. Close-up stereo view of FAD binding site of E. coli CPD photolyase in the cavity. Atomic coordinates for E. coli photolyase were obtained from the Protein Data Bank Žaccession number 1DNP.. This figure was drawn with the program Insight II ver. 97.0 ŽMolecular Simulation Source.. The polypeptide chain and FAD are shown as a gray ribbon; white, FAD binding residues.
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FAD binding sites indicate that similar reaction mechanisms exists in this family. Another feature of this protein family is the presence of a ‘C-terminal extension’ Žsee Fig. 3.. Although Class I CPD photolyase does not have any ‘C-terminal extension’, other groups contain various length of C-terminal extension except for dCRY w25,27–29x and S. alba CRY w14x and no amino acid sequence homology is found between any of the extensions. The C-terminal extension of Ž6-4.photolyases is relatively short Ž30–40 amino acids., while CRY have longer extension Žplant CRY 100–240 amino acids, animal CRY 100–160 amino acids.. Arabidopsis CRY 1 and 2 interact with phytochrome A Žredrfar-red photoreceptor in plant. through their C-terminal extension. Furthermore, the C-terminal extension is phosphorylated by phytochrome A-associated kinase activity following red light illumination w39x and this phosphorylation plays an important role for circadian rhythm in Arabidopsis. The C-terminal extension of animal CRY is also predicted to be a site for interaction with other proteins, but the functional role of the extension remains unknown. 2.3. Reaction mechanism In case of CPD photolyase, an electron is donated to CPD from the excited FADHy at the reaction center. The crystal structure of the E. coli CPD photolyase revealed key features of the reaction center w37x. The FAD chromophore lies deeply buried in the center of the helical domain, with direct access to solvent limited to a cavity leading from the edge of the isoalloxazine ring of FAD to the surface ŽFig. 4.. The cavity lies in the center of a tract of positive electrostatic potential that runs along the flat surface of the helical domain, and both the dimensions of the cavity and the asymmetric distribution of hydrophobic and polar residues within the cavity are appropriate to accommodate a CPD. These features led to the proposal that CPD photolyase binds DNA along the tract of positive electrostatic potential and that in the enzyme–substrate complex the CPD is flipped out of the DNA helix and into the cavity leading to FAD w37,40x. Once the cavity is occupied by a CPD, the adenine ring of the FADHy would be in van der Waals contact with CPD, consistent with the idea of
electron transfer between FADHy and CPD being important for catalysis. The high amino acid sequence homology between Ž6-4. and CPD photolyase suggested that Ž6-4.photolyase may have a structure and a reaction mechanism similar to those of CPD photolyase. In fact, Ž64.photolyase shows many properties analogous to CPD photolyase. Among several oxidative states of FAD, the fully reduced FAD ŽFADHy. is the active form in CPD photolyase w36,41x. When the FAD in CPD photolyase is oxidized at the purification step, the oxidized FAD in CPD photolyase can be photoreduced to fully reduced state by illuminating light in the presence of reducing agent. In this reaction Žphotoreduction. E-FADH8 abstracts an electron from W306 of CPD photolyase w42,43x. The photoreduction of oxidized FAD also occurs in Ž6-4.photolyase w44x and this tryptophan residue is conserved in all Ž6-4.photolyases w45x. Fully reduced FAD is the active form in Ž6-4.photolyase, indicating that electron donation from reduced FAD to the Ž6-4.photoproducts is very likely involved in the catalytic mechanism of Ž6-4.photolyase as well as CPD photolyase. Furthermore, protein modeling of Xenopus Ž6-4.photolyase using the E. coli CPD photolyase crystal structure as the starting point shows the presence of the cavity possessing FAD at the bottom ŽH. Nakamura, personal communication.. It is shown that on binding of Ž6-4.photolyase to the substrate DNA the Ž6-4.photoproduct flips out the DNA duplex w46x. Thus, it is very tempting to speculate that the flip out Ž6-4.photoproduct is captured into the hole where the electron is donated from FADH. In spite of these similarities, the reaction mechanism of Ž6-4.photolyase differs from that of CPD photolyase for the following reason. Although Ž64.photoproducts never revert to the canonical form by breakage of the sigma bond between C-6 of the 5X base and C-4 of the 3X base, direct analysis w44,47x and the mutagenic properties w48x of the repaired products show that Ž6-4.photolyase reverts Ž64.photoproducts to their native form. Furthermore, as described above, Ž6-4.photolyase repairs Ž6-4.photoproduct by electron donation as well as CPD photolyase w18,44,46x. These results indicate that structural alteration, which can be converted to the canonical form by electron donation, must occur prior to
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electron donation. A possible model for the repair mechanism of Ž6-4.photolyase has been proposed w18x. Ž6-4.photoproducts are formed using the oxetane species as intermediate from a dipyrimidine w16x Žsee Fig. 1.. The model proposes that Ž6-4.photolyase repairs the Ž6-4.photoproduct by first converting it to the oxetane intermediate on binding and subsequent electron donation to the oxetane intermediate results in the reversal to canonical dipyrimidines w18x. In the cavity, specific amino acid residues are proposed to interact with the groups at C-5 of the 5X base and at C-3 of the 3X base of Ž6-4.photoproducts to form the oxetane intermediate. Neither plant nor animal CRY shows any DNA repair activity although they have amino acid sequence homology with DNA photolyase, animal CRY have an especially high degree of homology with Ž6-4.photolyase. Protein modeling shows the presence of a putative cavity with FAD at the bottom in hCRY1 as well as in DNA photolyase ŽH. Nakamura, personal communication.. In the Drosophila cry b mutant a missense mutation was detected at Asp 410 in dcry gene w26x. This Asp residue is highly conserved among the DNA photolyaserblue light receptor family and has been shown to be involved in FAD binding in E. coli CPD photolyase ŽD 372 of E. coli CPD photolyase as shown in Fig. 4.. This indicates that FAD play an important role on CRY function. On the other hand, although only a missense mutation, the protein in this mutant is reduced at very low level while RNA level is reduced only slightly than wild type w26x. This might suggests that the CRY protein is very unstable in this mutant because of the mutation at the FAD binding site. These results suggest that CRY protein mediates signal transduction through electron donation, analogously to CPD photolyase w49x although the nature of the electron acceptor is not known. In plants, the two CRY proteins function in a distinct, but overlapping manner in various blue light responses or in response to differing light intensities w10–13,35x. In animals, the two CRY proteins might also have diverged functionally, since the two genes and proteins show different patterns of tissue-specific expression and cellular localization, mCRY1 but not mCRY2 being expressed strongly in SCN, while mCRY2 is expressed about three-fold high level than mCRY1 in the retina w30x. mCRY1 is localized in
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mitochondria whereas mCRY2 is found mainly in the nucleus w50x.
3. Conclusion Phylogenetic analysis suggests that an ancestral gene for this protein family encoded a CPD photolyase, which duplicated Žat least four times. before the divergence of prokaryotes and eukaryotes w45x. One of the copies then evolved to become Class II CPD photolyases in the eukaryotic lineage. Another copy evolved to be Class I CPD photolyase in the prokaryotic lineage. This copy Žor another copy. has been also transmitted to eukaryotes and become functionally divergent. It later became the Ž6-4.photolyase and animal CRY in animals, or the plant CRY in plants. For more than 2.5 billion years between the first appearance of living cells on the earth and the formation of ozone layer by release of oxygen from widespread cyanobacteria, living cells on the earth were continuously exposed to strong solar UV directly. Possessing an ability to overcome UV damage might have been essential for the cell to survive. Photolyase is the most simple and efficient repair system, and might have been the first DNA repair enzyme possessed by ancient organisms. Those organisms might have amplified photolyase genes by duplication many times. The amplified photolyase genes are the source for functionally divergent member of this protein family. This is a hypothesis which may answer our original question: What is the meaning of the structural relatedness of photolyase and cryptochrome? Our next question is: What is the functional role of the structural relatedness of photolyase and cryptochrome? The two types of protein of this family Žphotolyase and cryptochrome. are structurally related, although the phenomena in which the proteins are involved are seemingly different except for their both utilizing blue light. Both proteins contain FAD as chromophore, suggesting that they catalyze a light-dependent redox reaction. How can cryptochrome transduce signals by a redox reaction? This is the most important question to be solved in near future. Further studies on cryptochrome should continue to provide insights into the fundamental mechanisms of how organisms generally respond to light.
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Acknowledgements I apologize to all colleagues whose work has not been cited because of space limitations. I thank Mr. Kenichi Hitomi and Dr. Toshimi Mizukoshi for the schematic drawing of E. coli CPD photolyase ŽFig. 4. and Dr. Ciaren Morrison and Dr. Aziz Sancar for critical reading of this manuscript and for helpful comments. Studies in the RBC were supported by grants-in-aid for the Ministry of Education, Science, Sports and Culture of Japan.
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T. Todo r Mutation Research 434 (1999) 89–97 w28x C.P. Selby, A. Sancar, A third member of the photolyaserblue-light photoreceptor family in Drosophila: a putative circadian photoreceptor, Photochem. Photobiol. 69 Ž1999. 105–107. w29x S. Okano, S. Kanno, M. Takao, A.P.M. Eker, K. Isono, Y. Tsukahara, A. Yasui, A putative blue-light receptor from Drosophila melanogaster, Photochem. Photobiol. 69 Ž1999. 108. w30x Y. Miyamoto, A. Sancar, Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoreactive pigments for setting the circadian clock in mammals, Proc. Natl. Acad. Sci. USA 95 Ž1998. 6097–6102. w31x J.C. Hall, Tripping along the trail to the molecular mechanisms of biological clocks, Trends Neurosci. 18 Ž1995. 230– 240. w32x J.C. Dunlap, Molecular bases for circadian clocks, Cell 96 Ž1999. 271–290. w33x V. Suri, Z. Qian, J.C. Hall, M. Rosbash, Evidence that the TIM light response is relevant to light-induced phase shifts in Drosophila melanogaster, Neuron 21 Ž1998. 225–234. w34x R.J. Thresher, M.H. Vitaterna, Y. Miyamoto, A. Kazantsev, D.H. Hsu, C. Petit, C.P. Selby, L. Dawut, O. Smithies, J.S. Takahashi, A. Sancar, Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses, Science 282 Ž1998. 1490–1494. w35x D.E. Somers, P.F. Devlin, S.A. Kay, Phytochromes and crypthochromes in the entrainment of the Arabidopsis circadian clock, Science 282 Ž1998. 1488–1490. w36x G. Payne, A. Sancar, Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase, Biochemistry 29 Ž1990. 7715–7727. w37x H.-W. Park, S.-T. Kim, A. Sancar, J. Daisenhofer, Crystal structure of DNA photolyase from Escherichia coli, Science 268 Ž1995. 1866–1872. w38x C. Lin, D.E. Robertson, M. Ahmad, A.A. Raibekas, M. Schuman-Jorns, P.L. Dutton, A.R. Cashmore, Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1, Science 269 Ž1995. 968–970. w39x M. Ahmad, J.A. Jorillo, O. Smirnova, A.R. Cashmore, The
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Minireview
Functional diversity of the DNA photolyaserblue light receptor family Takeshi Todo
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Radiation Biology Center, Kyoto UniÕersity, Yoshida-konoe-cho, sakyoku, Kyoto 606-8501, Japan Accepted 16 March 1999
Keywords: DNA photolyase; CRY; DNA repair, UV damage; Circadian rhythm; Blue light photoreceptor; Cryptochrome
1. Introduction Proteins of the DNA photolyaserblue light photoreceptor family mediate either DNA repair or perceptionrsignal transduction of external light signals. Currently, this protein family is known to consist of three groups; cyclobutane pyrimidine dimer ŽCPD. photolyase, Ž6-4.photolyase and blue light photoreceptor Žcryptochrome: CRY.. Among them, CPD photolyase is the best characterized and its reaction mechanism has been elucidated in considerable detail. Compared with CPD photolyase, the reaction mechanisms of other two groups have been less well studied. The aim of this minireview is to summarize our current understanding of the characteristics of this protein family, focusing especially on the functional diversity and structural similarity between each group and CPD photolyase. Comparison between the structural and functional properties of these proteins will lead to understanding of each individual protein from a different viewpoint, especially by use of the wealth of the knowledge of the precise features of CPD photolyase.
) Tel.: q81-75-753-7560; Fax: q81-75-753-7564; E-mail: [email protected]
2. DNA photolyaser r blue light photoreceptor family 2.1. Functional diÕersity Photoreactivation ŽPR. reverses the effect of ultraviolet ŽUV. light when the organism is either concomitantly or subsequently exposed to blue light w1x. CPD and 6-4 wpyrimidine-2X-onex pyrimidines wŽ64.photoproductsx constitute major sources of base damage following the exposure of living cells to UV radiation w2x. Enzymes that are involved in the PR of UV-damaged DNA are called DNA photolyase w3–5x. DNA photolyase binds to UV-damaged DNA and, upon absorbing a blue light photon Žof wavelength 300 to 500 nm. splits CPD, restoring the bases to their native forms. Thus such photolyases are classified as CPD photolyases. CPD photolyase was first cloned from Escherichia coli in 1978 and subsequently the same gene was cloned from several organisms. CPD photolyases are classified into two classes by their primary structure: Class I and Class II CPD photolyases w6,7x. Class I CPD photolyase genes have been isolated from prokaryotes and eukaryotes Žfungi., but all from unicellular organisms w1,4,7x. Class II CPD photolyase genes have been
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isolated from a wide variety of organisms, including eubacteria, archaebacteria and higher eukaryotes w7– 9x. In the history of photolyase research, two new findings were made in 1993. One was the isolation of plant blue light photoreceptor genes that mediate numerous blue light-dependent responses w10x. The blue light photoreceptor gene was first identified as the causative gene for a mutant defective in a blue light sensing pathway mediating inhibition of hypocotyl elongation in Arabidopsis thaliana. This gene was named CRY 1, after cryptochrome, the name commonly given to plant blue-UVA photoreceptors. One more CRY gene Ž CRY 2 . was identified by similarity of its primarily structure to CRY 1 w11x. Mutation of this gene was shown to be responsible for later flowering than wild-type, thus regulating flowering time in response to light. In addition to the regulation of flowering time, CRY 2 shares considerable functional overlap with CRY 1. In seedling development, CRY 1 mediates high-intensity blue light responses and CRY 2 mediates low-light responses w12x. Furthermore, mutant plants lacking both CRY 1 and CRY 2 are deficient in the phototropic response w13x, although neither single mutant shows any phototropic defect. Thus, in plants, the CRY proteins function as photoreceptors for many blue light dependent responses. CRY gene was also isolated from Sinapis alba w14x. The apoproteins of Arabidopsis CRY 1 and 2 and Sinapis CRY are very similar to CPD photolyase and hence are structurally related to the photolyase, although CRY functions in signal transduction, not in DNA repair w15x.
The second was discovery of a new type of DNA photolyase, Ž6-4.photolyase. The CPD photolyase does not repair Ž6-4.photoproducts w1,3,4x. Because of the structural difference of a Ž6-4.photoproduct from a CPD w16x, it was thought that photoenzymatic reversal of Ž6-4.photoproducts was very unlikely for the following reason. The formation of Ž6-4.photoproduct involves the transfer of the hydroxy Žor amino. group at C-4 of the 3X base of the dinucleotide to the C-5 position of the 5X base concomitant with formation of a sigma bond between the C-6 of the 5X base and the C-4 of the 3X base ŽFig. 1.. Therefore, even if an enzyme breaks the 6-4 C–C bond, the bases would not be restored to their original forms. Thus, it had long been thought that Ž64.photoproducts were repaired exclusively by a light-independent repair mechanism, namely nucleotide excision repair. However, in 1993, it was reported that in Drosophila melanogaster there exists a light-dependent DNA repair activity specific for Ž6-4.photoproducts w17x. Later on, similar activity was further observed in some vertebrates and plants, but so far has not been detected in humans or in bacteria such as E. coli w17,18x. Currently, the cDNAs of Ž6-4.photolyase have been cloned from Drosophila w19x, Xenopus w20x, Arabidopsis w21x and Danio rerio ŽZebra fish. ŽY. Kobayashi, unpublished data.. The genes for the Ž6-4.photolyase apoprotein exhibit a sequence similarity to CPD photolyase, in particular to the Class I CPD photolyase w19x. Two genes having 41–45% sequence identity to Drosophila Ž6-4.photolyase are found in the human genome w19,22,23x. Since the proteins encoded by
Fig. 1. Reaction scheme for Ž6-4.photoproduct formation by UV irradiation. The proposed mechanism of Ž6-4.photolyase by the reverse reaction of formation is also shown by the arrow with dashed line.
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these genes does not show any DNA repair activity w22,24x and their functions have been predicted to be similar to those of plant cryptochrome, the proteins have been designated hCRY1 and hCRY2 w22x. A similar type of gene has also been identified in Drosophila and named as dcry w25–29x. These genes are expressed in a wide variety of tissues including inner tissues where light never reaches w19,22,23x. However, the murine homolog of hCRY, mCRY1 and mCRY2, are strongly expressed in suprachiasmatic nucleus ŽSCN. and the retina, respectively w30x, where the master clock and the photoreceptor for circadian rhythm are predicted to exist, respectively. Furthermore, in the former tissue mCRY1 expression oscillates with circadian rhythm w30x. This is the first finding suggesting the involvement of animal CRY in the circadian photoreceptor. Most organisms contain molecular time-keepers known as circadian clocks, which determine their daily biological rhythms w31,32x. Circadian clocks are set by the light and dark cycles of day and night, and once set keep time even in constant darkness. Exposure to light during the dark period can reset the clock w31,32x. Eyes are thought to be an input machinery to the clock, although eyeless and visually blind mutants of Drosophila exhibit normal light responses in circadian rhythm, suggesting that rhodopsins in eyes are not the circadian photoreceptor molecule w33x. In Drosophila, the light-induced phase shift of adult locomotor activity shows wavelength dependency in that maximal effects are observed with light of 400– 500 nm, while light of 600 nm or greater has no effects w33x. This action spectrum implicates the possible involvement of cryptochrome-like molecules in light response as a circadian photoreceptor, since the photolyasercryptochrome family comprises flavin-containing proteins having a characteristic absorption at 400–500 nm Žthe absorption maximum of an oxidated FAD-containing protein is 450 nm.. This possibility was demonstrated to be the case both in mouse w34x and Drosophila w25–27x. In mice, mutation of the cry gene has similar effects on circadian rhythm. Mice lacking mCRY2 were generated w34x. This knock-out mutant have reduced rate of photoinduction of a circadian reporter gene Ž per 1. in addition to the high amplitude phase shift in response to light pulses and the long free-running period w34x. A Drosophila mutant in the gene encod-
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ing dCRY was identified. In this mutant, circadian oscillation of clock gene Ž per and tim. expression disappeared w25x. Furthermore, the mutant shows no response to brief light pulses on resetting the clock Žphase shift of circadian rhythm. w26x. On the other hand, the phase shifts in transgenic flies over-expressing dCRY respond abnormally to light pulses w27x. More recently, it was reported that CRY 1 mutant of Arabidopsis shows the long free-running period under constant blue light w35x. These results indicate that CRY is a photoreceptor dedicated to the control of circadian rhythms. 2.2. Structural similarity Amino acid sequence alignment revealed that Class II CPD photolyase is distantly related to the other members of this family w7x. While members in each group of the protein family show relatively high mutual homology Ž25–80%., a much lower homology of 10–17% is found between each group except for that between Ž6-4.photolyase and animal CRY Ž40–60% homology.. Thus, this protein family can be classified into four groups from their primary structure: Ž1. Class I CPD photolyase, Ž2. Class II CPD photolyase, Ž3. plant CRY and Ž4. Ž6-4.photolyaseranimal CRY, although functionally they are divided into two groups: DNA repair ŽClass I and II CPD photolyase and Ž6-4.photolyase. and photoreceptor Žplant CRY and animal CRY. ŽFig. 2.. All known CPD photolyases contain two chromophores w3,4x. The first one is a reduced flavin adenine dinucleotide ŽFADHy. and second is either methenyltetrahydrofolate or deazaflavin w1,3,4x. The current consensus is that the second chromophore functions as an antenna which is elevated into an energetically excited state after absorbing blue light. This excited antenna molecule then transfers energy to FADHy, which in turn donates an electron to the CPD substrate held in the enzyme’s active site. This negative charge induces splitting of the cyclobutane ring of CPD. Subsequently, the electron is transferred back to flavin, restoring a CPD into its original state of two neighboring pyrimidines w3,4x. This repair reaction occurs even when the second chromophore is depleted from CPD photolyase w36x. In this case FADHy itself absorbs blue light. Thus, the FAD is necessary and sufficient for catalysis. Apoen-
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Fig. 2. Unrooted phylogenetic tree based on alignment of several members of the DNA photolyaserblue light receptor family. Abbreviations of species are: N. crassa ŽNc., S. cereÕisiae ŽSc., E. coli ŽEc., S. typhimurium ŽSt., A. nidurans ŽAn., H. halobium ŽHh., S. griseus ŽSg., A. thaliana ŽAt., S. alba ŽSa., C. reinhardtii ŽCr., M. domestica ŽMd., C. auratus ŽCa., D. melanogaster ŽDm., M. thermoautotrophicum ŽMt., Zebrafish ŽZ., H. sapiens Žh., X. laeÕis ŽXl..
zyme without FAD had no affinity for CPD but did regain its specific binding to CPD upon binding stoichiometrically to FAD w36x. This recovery of DNA binding activity by apophotolyase reconstituted with FAD indicates that this cofactor is structurally important in forming the DNA binding site. Thus, in addition to its essential role in catalysis, FAD also possesses an important role in the protein structure.
Structural features of FAD binding site of CPD photolyase have been analyzed precisely in E. coli CPD photolyase. The crystal structure of E. coli CPD photolyase was solved at a resolution of 2.3A w37x. The three-dimensional structure of CPD photolyase shows that the protein is folded into five b sheets and 20 a helices and these structural elements combine to form two domains, an N-terminal arb
Fig. 3. Domain structure of DNA photolyaserblue light receptor protein family. The domains of chromophore binding site and C-terminal extension are shown on the top. In the middle schematic diagram, conserved regions are shown by filled boxes.
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domain and a C-terminal helical domain. The flavin cofactor residues lies in the C-terminal helical domain and the folate chromophore is in the N-terminal domain ŽFig. 3.. The helical domain can be grouped into two clusters, Cluster I and Cluster II. Clusters I and II interact with the phosphate oxygens and riboflavin part of the FAD, respectively, indicating that the two helical clusters are kept together by binding with FAD to keep the higher structure of enzyme. All other member of this protein family also contain FAD as cofactor w20,22,24,28,29,38x. The second chromophore has not yet been characterized well, although a pterin is suggested to be a second chromophore in S. alba w15x, A. thaliana w13,15x and human CRY w22x. Regions highly conserved between
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all groups of the family exist at N-terminal and C-terminal half ŽHelical Domain. Žfilled box in Fig. 3.. The FAD binding sites identified in E. coli CPD photolyase exist in the highly conserved C-terminal half Žresidues 204–450.. The amino acid residues identified as FAD binding sites in E. coli CPD photolyase are shown in Fig. 4. The residues contacting with FAD are shown in white ŽTyr 222 , Thr 234 , Arg 236 , Leu237, Ser 238 , Trp 271 , Arg 278 , Trp 338 , Asn341, Asp 372 , Asp 374 and Asn378 .. These residues are conserved well among this protein family, although Tyr 222 is substituted by Phe in Ž6-4.photolyase, Arg 236 is substituted by Val in Ž6-4.photolyase, Trp 271 is substituted by Lys in Ž6-4.photolyase and animal CRY and N 341 is substituted by His in Ž6-4.photolyase and animal CRY. Conservation of
Fig. 4. Close-up stereo view of FAD binding site of E. coli CPD photolyase in the cavity. Atomic coordinates for E. coli photolyase were obtained from the Protein Data Bank Žaccession number 1DNP.. This figure was drawn with the program Insight II ver. 97.0 ŽMolecular Simulation Source.. The polypeptide chain and FAD are shown as a gray ribbon; white, FAD binding residues.
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FAD binding sites indicate that similar reaction mechanisms exists in this family. Another feature of this protein family is the presence of a ‘C-terminal extension’ Žsee Fig. 3.. Although Class I CPD photolyase does not have any ‘C-terminal extension’, other groups contain various length of C-terminal extension except for dCRY w25,27–29x and S. alba CRY w14x and no amino acid sequence homology is found between any of the extensions. The C-terminal extension of Ž6-4.photolyases is relatively short Ž30–40 amino acids., while CRY have longer extension Žplant CRY 100–240 amino acids, animal CRY 100–160 amino acids.. Arabidopsis CRY 1 and 2 interact with phytochrome A Žredrfar-red photoreceptor in plant. through their C-terminal extension. Furthermore, the C-terminal extension is phosphorylated by phytochrome A-associated kinase activity following red light illumination w39x and this phosphorylation plays an important role for circadian rhythm in Arabidopsis. The C-terminal extension of animal CRY is also predicted to be a site for interaction with other proteins, but the functional role of the extension remains unknown. 2.3. Reaction mechanism In case of CPD photolyase, an electron is donated to CPD from the excited FADHy at the reaction center. The crystal structure of the E. coli CPD photolyase revealed key features of the reaction center w37x. The FAD chromophore lies deeply buried in the center of the helical domain, with direct access to solvent limited to a cavity leading from the edge of the isoalloxazine ring of FAD to the surface ŽFig. 4.. The cavity lies in the center of a tract of positive electrostatic potential that runs along the flat surface of the helical domain, and both the dimensions of the cavity and the asymmetric distribution of hydrophobic and polar residues within the cavity are appropriate to accommodate a CPD. These features led to the proposal that CPD photolyase binds DNA along the tract of positive electrostatic potential and that in the enzyme–substrate complex the CPD is flipped out of the DNA helix and into the cavity leading to FAD w37,40x. Once the cavity is occupied by a CPD, the adenine ring of the FADHy would be in van der Waals contact with CPD, consistent with the idea of
electron transfer between FADHy and CPD being important for catalysis. The high amino acid sequence homology between Ž6-4. and CPD photolyase suggested that Ž6-4.photolyase may have a structure and a reaction mechanism similar to those of CPD photolyase. In fact, Ž64.photolyase shows many properties analogous to CPD photolyase. Among several oxidative states of FAD, the fully reduced FAD ŽFADHy. is the active form in CPD photolyase w36,41x. When the FAD in CPD photolyase is oxidized at the purification step, the oxidized FAD in CPD photolyase can be photoreduced to fully reduced state by illuminating light in the presence of reducing agent. In this reaction Žphotoreduction. E-FADH8 abstracts an electron from W306 of CPD photolyase w42,43x. The photoreduction of oxidized FAD also occurs in Ž6-4.photolyase w44x and this tryptophan residue is conserved in all Ž6-4.photolyases w45x. Fully reduced FAD is the active form in Ž6-4.photolyase, indicating that electron donation from reduced FAD to the Ž6-4.photoproducts is very likely involved in the catalytic mechanism of Ž6-4.photolyase as well as CPD photolyase. Furthermore, protein modeling of Xenopus Ž6-4.photolyase using the E. coli CPD photolyase crystal structure as the starting point shows the presence of the cavity possessing FAD at the bottom ŽH. Nakamura, personal communication.. It is shown that on binding of Ž6-4.photolyase to the substrate DNA the Ž6-4.photoproduct flips out the DNA duplex w46x. Thus, it is very tempting to speculate that the flip out Ž6-4.photoproduct is captured into the hole where the electron is donated from FADH. In spite of these similarities, the reaction mechanism of Ž6-4.photolyase differs from that of CPD photolyase for the following reason. Although Ž64.photoproducts never revert to the canonical form by breakage of the sigma bond between C-6 of the 5X base and C-4 of the 3X base, direct analysis w44,47x and the mutagenic properties w48x of the repaired products show that Ž6-4.photolyase reverts Ž64.photoproducts to their native form. Furthermore, as described above, Ž6-4.photolyase repairs Ž6-4.photoproduct by electron donation as well as CPD photolyase w18,44,46x. These results indicate that structural alteration, which can be converted to the canonical form by electron donation, must occur prior to
T. Todo r Mutation Research 434 (1999) 89–97
electron donation. A possible model for the repair mechanism of Ž6-4.photolyase has been proposed w18x. Ž6-4.photoproducts are formed using the oxetane species as intermediate from a dipyrimidine w16x Žsee Fig. 1.. The model proposes that Ž6-4.photolyase repairs the Ž6-4.photoproduct by first converting it to the oxetane intermediate on binding and subsequent electron donation to the oxetane intermediate results in the reversal to canonical dipyrimidines w18x. In the cavity, specific amino acid residues are proposed to interact with the groups at C-5 of the 5X base and at C-3 of the 3X base of Ž6-4.photoproducts to form the oxetane intermediate. Neither plant nor animal CRY shows any DNA repair activity although they have amino acid sequence homology with DNA photolyase, animal CRY have an especially high degree of homology with Ž6-4.photolyase. Protein modeling shows the presence of a putative cavity with FAD at the bottom in hCRY1 as well as in DNA photolyase ŽH. Nakamura, personal communication.. In the Drosophila cry b mutant a missense mutation was detected at Asp 410 in dcry gene w26x. This Asp residue is highly conserved among the DNA photolyaserblue light receptor family and has been shown to be involved in FAD binding in E. coli CPD photolyase ŽD 372 of E. coli CPD photolyase as shown in Fig. 4.. This indicates that FAD play an important role on CRY function. On the other hand, although only a missense mutation, the protein in this mutant is reduced at very low level while RNA level is reduced only slightly than wild type w26x. This might suggests that the CRY protein is very unstable in this mutant because of the mutation at the FAD binding site. These results suggest that CRY protein mediates signal transduction through electron donation, analogously to CPD photolyase w49x although the nature of the electron acceptor is not known. In plants, the two CRY proteins function in a distinct, but overlapping manner in various blue light responses or in response to differing light intensities w10–13,35x. In animals, the two CRY proteins might also have diverged functionally, since the two genes and proteins show different patterns of tissue-specific expression and cellular localization, mCRY1 but not mCRY2 being expressed strongly in SCN, while mCRY2 is expressed about three-fold high level than mCRY1 in the retina w30x. mCRY1 is localized in
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mitochondria whereas mCRY2 is found mainly in the nucleus w50x.
3. Conclusion Phylogenetic analysis suggests that an ancestral gene for this protein family encoded a CPD photolyase, which duplicated Žat least four times. before the divergence of prokaryotes and eukaryotes w45x. One of the copies then evolved to become Class II CPD photolyases in the eukaryotic lineage. Another copy evolved to be Class I CPD photolyase in the prokaryotic lineage. This copy Žor another copy. has been also transmitted to eukaryotes and become functionally divergent. It later became the Ž6-4.photolyase and animal CRY in animals, or the plant CRY in plants. For more than 2.5 billion years between the first appearance of living cells on the earth and the formation of ozone layer by release of oxygen from widespread cyanobacteria, living cells on the earth were continuously exposed to strong solar UV directly. Possessing an ability to overcome UV damage might have been essential for the cell to survive. Photolyase is the most simple and efficient repair system, and might have been the first DNA repair enzyme possessed by ancient organisms. Those organisms might have amplified photolyase genes by duplication many times. The amplified photolyase genes are the source for functionally divergent member of this protein family. This is a hypothesis which may answer our original question: What is the meaning of the structural relatedness of photolyase and cryptochrome? Our next question is: What is the functional role of the structural relatedness of photolyase and cryptochrome? The two types of protein of this family Žphotolyase and cryptochrome. are structurally related, although the phenomena in which the proteins are involved are seemingly different except for their both utilizing blue light. Both proteins contain FAD as chromophore, suggesting that they catalyze a light-dependent redox reaction. How can cryptochrome transduce signals by a redox reaction? This is the most important question to be solved in near future. Further studies on cryptochrome should continue to provide insights into the fundamental mechanisms of how organisms generally respond to light.
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Acknowledgements I apologize to all colleagues whose work has not been cited because of space limitations. I thank Mr. Kenichi Hitomi and Dr. Toshimi Mizukoshi for the schematic drawing of E. coli CPD photolyase ŽFig. 4. and Dr. Ciaren Morrison and Dr. Aziz Sancar for critical reading of this manuscript and for helpful comments. Studies in the RBC were supported by grants-in-aid for the Ministry of Education, Science, Sports and Culture of Japan.
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