Photolyase/Cryptochrome Blue-Light Photoreceptors Use Photon Energy to Repair DNA and Reset the Circadian Clock

Photolyase/cryptochrome blue-light photoreceptors use photon energy to repair DNA and reset the circadian clock

Carol L Thompson1 and Aziz Sancar*,1

1Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, NC 27599, USA

Blue light governs a number of cellular responses in and mutagenic effects of far-UV are reversed by bacteria, plants, and animals, including photoreactiva- irradiation with near-UV/blue-light (300 – 500 nm) in tion, plant development, and circadian photoentrainment. a process called photoreactivation, which is catalyzed These activities are mediated by a family of highly by photolyase. conserved flavoproteins, the photolyase/cryptochrome Cryptochrome mediates a separate blue-light depen- family. Photolyase binds to UV photoproducts in DNA dent function, circadian photoreception, which resets and repairs them in a process called photoreactivation in the circadian clock to the solar day. The circadian which blue light is used to initiate a cyclic electron clock is an intrinsic timekeeping system in many transfer to break bonds and restore the integrity of organisms, which regulates oscillations in behavior DNA. Cryptochrome, which has a high degree of and physiological processes, such as sleep cycles and sequence identity to photolyase, works as the main metabolic rate. Circadian rhythms have a periodicity of circadian photoreceptor and as a component of the about 24 h (‘circa’=about; ‘dies’=day) and exist even molecular clock in animals, including mammals, and in the absence of light stimulus. Cryptochromes sense regulates growth and development in plants. blue light and synchronize an organism’s behavior with Oncogene (2002) 21, 9043 – 9056. doi:10.1038/sj.onc. the light cycle, but the mechanism of phototransduc- 1205958 tion is unknown. The photolyase/cryptochrome family consists of Keywords: pyrimidine dimers; DNA photorepair; 55 – 70 kD monomeric proteins that contain two circadian clock; photosensory pigment non-covalently bound prosthetic groups, flavin adeno- nucleotide (FAD) and a pterin (or in rare cases, a deazaflavin), and all members of this family share a Introduction high degree of sequence identity within their FAD- binding domains (Todo, 1999; Deisenhofer, 2000; In organisms ranging from bacteria to mammals, a Sancar, 2000). Photolyases possess a positively myriad of responses to blue light, including photo- charged groove that binds the phosphodiester back- reactivation and circadian entrainment, are mediated bone of DNA and a pocket for binding either by one family of highly homologous proteins. The cyclobutane pyrimidine dimers or (6 – 4) photopro- photolyase/cryptochrome family is comprised of two ducts. The sequence homology between cryptochrome types of proteins that carry out distinct functions: and photolyase in these regions suggests that both photolyase, which harnesses blue-light energy to break enzymes share common structure and chromophore bonds and repair UV photoproducts in DNA; and binding and may share a similar mechanism of action, cryptochrome, which acts as a sensor of environmental although cryptochrome has no demonstrable DNA light to regulate processes ranging from circadian repair activity. Cryptochrome possesses an additional entrainment in animals and plants, to plant growth C-terminal tail that extends past the region of and development. homology with photolyase and is believed to be Photolyase is a blue-light activated DNA repair involved in signal transduction and protein – protein enzyme. Far-UV (200 – 300 nm) light induces forma- interaction. The cyclic electron transfer mechanism tion of photoproducts such as cyclobutane pyrimidine utilized by photolyase has been well characterized and dimers (Pyr54Pyr) and pyrimidine-pyrimidone 6-4 may prove to be a basis for uncovering the photoproducts (Pyr [6-4] Pyr), whose deleterious effects mechanism behind cryptochrome-mediated photo- include inhibition of replication and transcription, transduction. mutation, growth arrest and cell death. The lethal Photolyase family members have no obvious sequence homology to other classes of flavoproteins, perhaps because activated photolyase utilizes flavin in its light-excited state as opposed to flavin oxidoreduc- *Correspondence: A Sancar, Department of Biochemistry and Biophysics, CB #7260, University of North Carolina School of tase which uses ground-state flavin. Photolyase has Medicine, Chapel Hill, NC 27599-7260, USA; persisted throughout evolution and is found among E-mail: [email protected] animals, plants, and bacteria (Table 1). Photolyase is Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9044 Table 1 Photolyase and cryptochrome in different kingdoms Species Kingdom Photolyase (6-4)photolyase Cryptochrome

E. coli Eubacteria + 77 B. subtilis Eubacteria 77 7 B. firmis Eubacteria + 77 V. cholerae Eubacteria + 7 +(2?) Synechocystis sp. Eubacteria + 7 +(?) Mesorhizobium loti Eubacteria + 77 M. thermoautotrophicum Archaea + 77 M. Jannaschii Archaea 77 7 Halobacterium sp. Archaea + 7 +(?) S. cerevisiae Eukarya + 77 S. pombe Eukarya 77 7 C. elegans Eukarya 77 7 D. melanogaster Eukarya + + + X. laevis Eukarya + + +(3) H. sapiens Eukarya 77+(2) A. thaliana Eukarya + + +(2) D. rerio Eukarya ? + +(6) G. gallus Eukarya 77+(2)

found in organisms as ancient as archaebacteria, and a Repair of this adduct cannot be achieved by breaking photolyase gene is even found in certain animal viruses the (6 – 4) C-C bond. The (6 – 4) photolyase presumably (Afonso et al., 1999, 2000; Willer et al., 1999; stabilizes the oxetane intermediate which is then split Srinivasan et al., 2001). Cryptochrome has only been by electron transfer to restore the bases. identified thus far in plants and animals, excluding C. elegans, although putative homologs have been found Cryptochrome ‘Cryptochrome’ was originally coined in bacteria (Hitomi et al., 2000; Ng and Pakrasi, 2001). as a generic term to describe plant blue-light receptors While the marsupials have retained both photolyase which were known to exist but had not been identified and cryptochrome during evolution, placental for nearly a century. Now the term is used to designate mammals only have the cryptochrome, and in the photolyase sequence homologs with no DNA repair absence of photolyase, UV photoproducts in these function but with known or presumed blue-light latter organisms are removed by nucleotide excision receptor function (Sancar, 2000). In plants, crypto- repair exclusively (Sancar, 1996; Wood, 1997). With chromes regulate a variety of growth processes in the diversity of organisms that contain these proteins, response to blue-light, and are a significant topic of the photolyase/cryptochrome family has been classified research in plant biology. However, interest in by three different criteria: function, chromophore cryptochromes has dramatically escalated since our composition, and sequence homology. proposal that these pigments are the primary circadian photoreceptor in mammals and possibly in other organisms (Hsu et al., 1996; Zhao and Sancar, 1997; Classification Miyamoto and Sancar, 1998). When classified by function, there are three distinct Photolyase family members may also be classified by categories. chromophore composition. All photolyases and cryptochromes non-covalently bind the primary Cyclobutane pyrimidine dimer (CPD) photolyase Ap- chromophore, flavin, within a pocket buried in the proximately 70 – 80% of DNA photoproducts folded enzyme. The ‘second chromophore’, as it is generated by ultraviolet light are cis,syn-cyclobutane called, acts as a photoantenna and transfers excitation pyrimidine dimers, where a cyclobutane ring is formed energy to the catalytic FADH7 cofactor by fluores- between the C5 and C6 carbons of each base. CPD cence resonance energy transfer (FRET). The majority photolyase, referred to as simply ‘photolyase’, utilizes a of photolyases, all known (6 – 4) photolyases, and cyclic electron transfer to break this bond, resulting in cryptochromes (Malhotra et al., 1995), contain a folate restoration of the natural bases. (MTHF) as the second chromophore, referred to as the ‘Folate Class’ (Sancar and Sancar, 1987, 1988). The (6 – 4) photoproduct photolyase The CPD photolyase ‘Deazaflavin Class’ represents a handful of CPD cannot repair this lesion. A specific enzyme called (6 – photolyases and cryptochromes from those few species 4) photolyase specifically binds to and repairs the (6 – which can synthesize the ‘ancient molecule’ 8-HDH, 4) photoproduct (Todo et al., 1993; Kim et al., 1994). including Streptomyces griseus, Anacystis nidulans, and The pyrimidine – pyrimidone (6 – 4) photoproduct, Methanobacterium thermoautotrophicum (Eker et al., which comprises 20 – 30% of total UV-induced photo- 1988; Kiener et al., 1989). products, is formed through an oxetane intermediate to On the basis of sequence homology, two classes join C6 of the 5’ base to the C4 of the 3’ base, with the were proposed to exist in the photolyase/crypto- addition of a hydroxyl group at the C5 of the 5’ base. chrome family which are independent of both

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9045

Figure 1 Sequence alignment of Type 1 photolyase family members. Cryptochromes Cry1 and Cry2 from mouse, (6 – 4) photolyase from Drosophila melanogaster, E. coli photolyase, and Arabidopsis Cry2 are shown. Colors: yellow, identical; blue, most frequent; pink, strongly similar; green, weakly similar

Figure 2 Crystal structure of E. coli photolyase. The ribbon diagram representation (left) shows the orientation of the two chromophores. In the surface potential representation (right) the DNA binding groove is visible as a blue groove along the protein and the thymidine dimer binding pocket is indicated by a dashed rectangle

function and chromophore composition, Type I and and plants, as well as some microbial photolyases Type II (Kato et al., 1994; Yasui et al., 1994). Type I (Todo, 1999). However, as more gene sequences of photolyases include most of the microbial CPD photolyases and cryptochromes become available photolyases, all (6 – 4) photolyases and cryptochromes demonstrating the existence of more sequence groups, (Kanai et al., 1997) (Figure 1), while Type II the utility of Type I and II classiﬁcation has become photolyases include CPD photolyases from animals limited.

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9046 Photolyase Spectral properties of photolyase The composition of the second chromophore affects Crystal structure both the spectral properties and activity of the enzyme. Examination of the crystal structure of photolyase has Photolyases are chromoproteins because of their clarified the mechanism of photoreactivation (Figure 2) absorbance in the 300 – 500 nm range, due primarily (Deisenhofer, 2000; Sancar, 2000). Crystal structures, to the presence of the folate, with some contribution of available for the CPD photolyases from E. coli (Park et the FAD. Although photolyase containing only the al., 1995), A. nidulans (Tamada et al., 1997), and T. FADH7 (the active form of FAD in photolyase) and thermophilus (Komori et al., 2001), revealed that no second chromophore is functional, presence of the photolyase consists of two domains: an N-terminal a/ folate or deazaflavin increases efficiency 5 – 10-fold and b domain and a C-terminal helical domain, connected dominates the absorption spectrum (Figure 3a,b). by an interdomain loop. The a/b domain consists of a While folate alone has peak absorbance at 360 nm, b-sheet with five parallel strands and five a-helices in a the folate class of enzymes has absorbance maxima structure typical of a dinucleotide binding fold. The ranging from 377 nm for S. cerevisiae photolyase helical domain consists of 14 helices that form a flat (short wavelength photolyase) (Sancar et al., 1987a) face surrounding a hole leading into the center of to 410 nm for Bacillus firmus (medium wavelength protein where the FAD is buried and held in place by photolyase) (Malhotra et al., 1994). The 15 – 50 nm contacts with 14 conserved amino acids. The interac- red-shift of the folate absorption is due to constraints tion between the domains is predominantly polar, and imposed upon the chromophore by binding to the in E. coli MTHF binds to the exterior of the shallow enzyme (Johnson et al., 1988). Deazaflavin-containing cleft formed between the domains. For T. thermophilus photolyases represent the long wavelength class of and A. nidulans, the cleft is large enough for the photolyases, with an absorbance maximum of 440 nm smaller 8-HDF cofactor to bind in the interior of the for A. nidulans, S. griseus, and M. thermoautrophicum enzyme. The distance between the centers of the two photolyases (Eker et al., 1981, 1988; Kiener et al., cofactors is 16.8 A˚ in E. coli photolyase and 17.7 A˚ in 1989), a 20 nm red-shift from native deazaflavin the A. nidulans enzyme. Based on the structure it is absorbance (420 nm). The deazaflavin-containing proposed that the phosphodiester backbone of DNA enzymes exhibit a higher absolute quantum yield than binds photolyase on the flat surface of the helical the folate-containing enzymes, due to the higher domain along a trace of positive electrostatic potential. efficiency of energy transfer to the flavin. To date, Mutation of these positively charged residues results in accurate spectral studies of either (6 – 4) photolyase or a drastic reduction of substrate binding (Baer and cryptochrome are not available because active purified Sancar, 1993). The thymidine dimer is proposed to ‘flip enzymes from native sources are not available, out’ from the DNA helix into the central cavity where although preliminary absorption spectra of recombi- it comes within van der Waals contact distance of the nant proteins expressed in E. coli with FAD molecule (Park et al., 1995). The pocket is lined substoichiometric amounts of both chromophores with hydrophobic residues on one side and polar exhibit peak absorption at 420 nm (Figure 3c,d) residues on the other which matches the asymmetric (Malhotra et al., 1995; Zhao et al., 1997). polarity of the thymidine dimer. Support for ‘base- The oxidation state of the flavin is key to its activity. flipping’ comes from the co-crystal of T. thermophilus During purification, the FADH7 cofactor often with thymine, which reveals the binding of thymine becomes oxidized to yield either the flavin neutral within the inner cavity of the protein (Komori et al., radical (FADH8) or FADox. Photolyase enzyme 2001). preparations which are blue in color contain the While crystal structures are not available for (6-4) neutral radical form of flavin because of its strong photolyase or for cryptochromes, there is a high degree absorbance at the longer wavelengths 580 and 625 nm of conservation throughout the FAD chromophore as well as absorbance at 380 and 480 nm (Jorns et al., binding region and the DNA binding region. Mole- 1984). The active ground state of photolyase is the cular modeling of (6 – 4) photolyase indicates the two-electron reduced form. The neutral radical is presence of an active site pocket large enough to inactive and it must be reduced to activate the enzyme accommodate a (6 – 4) photoproduct (Todo, 1999), and in vitro. However, in vivo the flavin is always in the molecular modeling of cryptochrome on the a-back- two-electron reduced FADH7 state (Payne et al., bone of the E. coli photolyase results in a well- 1987). Whether or not a fully reduced flavin is conserved structure, although cryptochrome possesses necessary for cryptochrome is not known at present. an additional C-terminal tail not found in photolyase. Furthermore, crystal structures for both folate class Reaction mechanism of photolyase and deazaflavin class photolyases are nearly super- imposable (Tamada et al., 1997), indicating that all Photolyase (and cryptochromes) possess both light- photolyase family members may have the same basic independent and light-dependent functions. In the case architecture. While this structural conservation is of photolyase, substrate binding is performed in a expected for (6 – 4) photolyase, the implications for light-independent fashion, and the enzyme either cryptochrome are as yet unknown. remains bound until it is exposed to photoreactivating

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9047

Figure 3 Absorption and action spectra of photolyase/cryptochrome family. (a) Absorption spectra of E-FADH7 MTHF7 (solid line) and E-FADH7 (dashed line) forms of E. coli photolyase. The squares and triangles are the values for quantum yield 6 molar extinction coefficient, e 6 j, superimposed on the absolute absorption spectrum (Payne and Sancar, 1990). (b) Absorption of for E- FADH7 8DF (solid line) and the E-FADH7 (dashed line) forms of A. nidulans photolyase. Circles and triangles are values for e 6 j (Malhotra et al., 1992). (c) Absorption of recombinant D. melanogaster (6 – 4) photolyase and (d) absorption of recombinant human cryptochome 1 with substoichiometric amounts of chromophores light and catalyzes the repair reaction, or it stimulates unaffected by light, and no physical interaction nucleotide excision repair of UV-induced photopro- between photolyase and the excision repair machinery ducts in the dark. Nucleotide excision repair is a has been found. general DNA repair mechanism which is based on the The light-dependent function of photolyase, photo- ability of the excision nuclease to recognize bulky reactivation, has been well characterized for lesions that distort the DNA helix (Sancar, 1996). cyclobutane pyrimidine dimer repair (Sancar, 1994) Photolyase, in binding to photoproducts and ‘flipping (Figure 4a). After light-independent binding of its out’ the damaged dinucleotide, increases distortion of substrate through three-dimensional diffusion with the DNA helix at the damage site and thereby low-specificity recognition mediated primarily by ionic accelerates damage recognition and assembly of the interactions with the backbone of the damaged DNA excision repair complex (Sancar et al., 1984; Hays et strand, the dimer is ‘flipped out’ into the center cavity al., 1985; Sancar and Smith, 1989). In addition to UV of the protein to bring it into proximity with the photoproducts, E. coli photolyase was able to bind to flavin, and forms a high-specificity, high-affinity cisplatin adducts with only a 10-fold decrease in affinity enzyme-substrate complex. The folate (or deazaflavin) compared to cyclobutane dimers, UvrABC-mediated acts as a ‘photoantenna’ to absorb a photon of 300 – excision repair of cisplatin adducts was stimulated by 500 nm light, and achieves a high-energy excited 1.3-fold, and photolyase containing cells were more singlet state with a lifetime of 0.5 ns (MTHF) or resistant to cisplatin than cells lacking photolyase 2.0 ns (8-HDF). Fluorescence resonance energy trans- (O¨ zer et al., 1995). The detailed mechanism of fer occurs from the folate to the flavin (FADH7) with stimulation of excision repair is unknown; it is a quantum yield of 0.8. Deazaflavin-containing

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9048

Figure 4 Photoreactivation mechanism. (a) Photolyase. After absorption of a photon by folate, energy is transferred to ﬂavin, which donates an electron to break the cyclobutane ring. (b) (6 – 4) photolyase. (6 – 4) photolyase thermally converts (6 – 4) photoproduct to an oxetane intermediate, and then initiates photoinduced cyclic electron transfer to break the oxetane ring

photolyases are more efficient than the Folate Class, any net change in the chromophores or in the protein. with energy transfer from deazaflavin to flavin The (6-4) photolyase is believed to function by a occurring with a quantum yield of 0.98. Electron similar mechanism with one significant difference. transfer occurs from the flavin to the Pyr54Pyr with Upon binding, photolyase first thermally converts a quantum yield of 0.9 – 0.95. The cyclobutane ring is the (6-4) photoproduct to an oxetane intermediate, split by bond rearrangement in the Pyr54Pyr radical which resembles the cyclobutane ring, and photo- anion. Lastly, back electron transfer to FADH8 induced electron transfer breaks the ring and restores regenerates the active form of flavin, FADH7, the bases (Figure 4b) (Kim et al., 1994; Todo et al., completing the cyclic electron transfer cycle without 1996; Zhao et al., 1997).

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9049 expression of the C-terminus alone was sufficient to Cryptochrome cause a constitutive light response in plants (Yang et al., 2000). These results have led to the following Reaction mechanism model: cryptochrome in the ground state represses The high degree of sequence identity and structural signaling by the C-terminus; upon blue-light activa- conservation between cryptochrome and photolyase tion, the C-terminus is released from its repressed suggests that cryptochrome utilizes a similar reaction state to initiate signal transduction through protein – mechanism: after light-independent binding of a protein interactions. substrate, blue light initiates a cyclic electron transfer Two responses to light have been observed in plant reaction. Cryptochrome has not been shown to employ cryptochromes. First, CRY2, but not CRY1, is rapidly any of the typical signaling methods used by other degraded by blue-light through an unknown mechan- sensory proteins: protein conformational change, ism (Lin et al., 1998). Second, both CRY1 and CRY2 quaternary structure changes, G protein activation, or are phosphorylated in the C-terminal domain in phosphorylation. It is possible that cryptochrome response to either blue- or red-light (Ma´ s et al., 2000; combines one or more of these methods with electron Ahmad et al., 1998; Shalitin et al., 2002). Although the transfer, such as a photoinduced electron transfer significance of this phosphorylation event is unknown, resulting in a conformational change in the C-terminus the existence of this interaction indicates that cross-talk that activates it for signaling. However, the substrate is between photoreceptive systems can be mediated unknown, as is the signal transduction mechanism that directly between photoreceptors, and this physical results in resetting the circadian clock by light. relationship between two different photosensory Although the biochemical data for a photocycle is proteins, cryptochrome and phytochrome, as well as lacking, there is strong genetic evidence for crypto- the existence of yet another class of plant blue-light chrome as a circadian photoreceptor in plants and photoreceptors, phototropins (Briggs and Huala, 1999; animals. Elich and Chory, 1997), has complicated the study of the role of cryptochromes in plants. Cryptochromes in plants Cryptochromes in animals Cryptochrome was first discovered as a possible photoreceptor in Arabidopsis thaliana when it was Animal cryptochromes were first discovered in 1995, cloned as the gene complementing the hy4 photo- when the examination of the human genome revealed morphogenic mutant which has impaired blue-light a putative photolyase homolog (Adams et al., 1995). dependent inhibition of hypocotyl elongation (Ahmad Although human cells had been shown to lack and Cashmore, 1993). With 30% sequence identity to photoreactivation (Li et al., 1993), the homology of microbial photolyase, a blue-light activated enzyme, it a particular EST to photolyase was so strong that it appeared to be a good candidate for a blue-light was unclear if this gene might still encode a functional photoreceptor and was named CRY1. A second gene, photolyase. Both this first homolog and a second CRY2, has since been cloned in A. thaliana (Hoffman human homolog discovered later (Hsu et al., 1996) et al., 1996; Lin et al., 1998) as well as cryptochromes were expressed in heterologous systems and were from other plants including Sinapis alba (mustard). tested for photolyase activity and found to have no Like photolyase, recombinant plant cryptochromes repair activity on either cyclobutane dimers or (6 – 4) contain FAD (Malhotra et al., 1995; Lin et al., 1995) photoproducts (Hsu et al., 1996; Todo et al., 1997). and folate (Malhotra et al., 1995), but these proteins Because of the precedence for photolyase homologs had no demonstrable photolyase activity (Malhotra et functioning as blue-light photoreceptors in plants, we al., 1995; Lin et al., 1995). proposed that these human homologs might be blue- Genetic analysis of CRY1 and CRY2 Arabidopsis light photoreceptors in mammals (Hsu et al., 1996) mutants implicated both genes in blue-light inhibition although mammalian cryptochromes are more closely of hypocotyl elongation (Ahmad and Cashmore, 1993; related to (6-4) photolyase than to plant crypto- Lin et al., 1998; Guo et al., 1998), flowering time chromes (Figure 1). Cryptochromes are widespread in regulation (Guo et al., 1998), and, following the the animal kingdom, with the notable exception of C. discovery of the role of CRY in the mammalian elegans (Table 1). Humans and mice each have two circadian clock, CRY1 was also implicated in cryptochromes, named hCry1 and hCry2 or mCry1 circadian clock control (Somers et al., 1998). Trans- and mCry2 (Hsu et al., 1996). While Drosophila only genic plants overexpressing plant cryptochromes has a single cryptochrome (Ishikawa et al., 1999; Egan exhibited enhanced sensitivity to blue-light in terms et al., 1999; Selby and Sancar, 1999), Danio rerio of hypocotyl elongation (Lin et al., 1996, 1998). (zebrafish) has up to six cryptochrome homologs Perhaps the most intriguing result of genetic studies (Kobayashi et al., 2000). Although cryptochrome is of cryptochromes in plants comes from the creation of generally accepted as a circadian photoreceptor in transgenic Arabidopsis plants that overexpress the C- Drosophila, in mice the cryptochromes are integrated terminal domain of CRY1 and CRY2. The C- into the transcriptional feedback loop of the circadian terminus of cryptochrome is postulated to be the clock making separation of its clock and photorecep- mediator of signal transduction; accordingly, over- tive functions difficult.

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9050

Figure 5 Expression of mouse cryptochromes in retina and SCN. (a) Retina. Arrows show regions of high expression of cryptochromes in the inner retina. Expression of opsin is shown for comparison. Abbreviations: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (b) SCN. Cry1 but not Cry2 is strongly expressed in the suprachiasmatic nucleus (arrows)

Tissue expression in mammals present (Figure 5a), and led us to conclude that CRYs Cryptochromes are predominantly nuclear proteins are circadian photoreceptors (Miyamoto and Sancar, (Petit and Sancar, 1999) expressed in virtually all cell 1998; Thresher et al., 1998). Recently a photoreceptive types (Miyamoto and Sancar, 1998), consistent with net with a distinct morphology comprising 1% of the fact that all cells possess autonomous circadian retinal ganglion cells has been found with direct clocks (Balsalobre et al., 1998). Cry1 is highly projections to the suprachiasmatic nucleus (Berson et expressed in the liver and testes and Cry2 is highly al., 2002; Provencio et al., 2002). These cells are expressed in brain (Miyamoto and Sancar, 1998, 1999). directly photosensitive, as measured by whole cell However, some tissues, such as the retina and the current clamp recordings, and this photoresponse has suprachiasmatic nucleus, exhibit higher expression been attributed to melanopsin (Hattar et al., 2002), a levels than others, perhaps indicative of their speciﬁc candidate photoreceptor which is expressed in these functions in these locations (Miyamoto and Sancar, cells and has homology to invertebrate opsin (Proven- 1998). cio et al., 2000). However, melanopsin knockout mice The retina is the sole site of circadian photoreception have normal circadian photoreception, and no data in mammals (Roenneberg and Foster, 1997). Mice with exists as of yet to support the role of melanopsin as a certain retinal degenerative diseases lose their visual photoreceptor. The presence of cryptochrome in the photoreceptors but possess an intact inner retina; these ganglion cell layer is supportive of a role for mice retain circadian photosensitivity, indicating that cryptochrome in this photoreceptive net. visual photoreceptors are unnecessary and that a The suprachiasmatic nucleus (SCN) is a structure in circadian photoreceptor resides in the inner retina. the hypothalamus that acts as a master regulator of the Examination of mCry1 and mCry2 expression in the circadian clock. We found that mCry1 mRNA is retina revealed high expression of cryptochromes in expressed at a high level in the SCN, and moreover, this region, in both the ganglion cell layer and the the mRNA oscillates with circadian rhythmicity, one of inner nuclear layer, with slightly higher levels of mCry2 the ﬁrst indications of a role for cryptochromes in

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9051 constant darkness, mice became arrhythmic (Figure 6d) (van der Horst et al., 1999; Vitaterna et al., 1999). The essential role of cryptochromes in the circadian clock made further analysis of the role of cryptochromes in circadian photoreception by behavioral studies impos- sible in these mice. Another quantifiable photoresponse, acute gene induction in the SCN by light, is a marker of phototransduction to the SCN and is an initial event in clock resetting by light (Shigeyoshi et al., 1997). Single Cry knockouts exhibited blunted mPer1 gene induction in the SCN by light (Thresher et al., 1998; Vitaterna et al., 1999). The cry17/7cry27/ 7 mice lost mPer1 induction entirely, exhibiting an elevated basal level of mPer1 in the dark, consistent with Crys being photoreceptors and components of the molecular clock. However, mPer2 mRNA remained inducible by light in these animals, leading to the conclusion that cryptochromes were not the sole photoreceptors mediating phototransduction to the SCN (Vitaterna et al., 1999). Other mouse models have also contributed to the understanding of the role of cryptochromes in the circadian clock. While cryptochrome double-knockout mice still respond to light molecularly and behaviorally, we found that mice without cryptochromes and without visual photoreceptors lose all behavioral photoresponses and have severely reduced fos induc- Figure 6 Wheel-running actograms of cryptochrome mutants. tion by light in the SCN (Figure 7) (Selby et al., 2000), Circadian behavior of knockout mice was measured first during 12 : 12 h light : dark cycles, shown by the bar above each acto- the first evidence that classical opsins play a redundant gram: black, dark; white, light. Animals then entered constant role with cryptochromes in modulating light-driven darkness (arrow), where the intrinsic circadian period can be mea- responses. Lastly, when mice mutant for plasma retinol sured without light input to the clock. Data for each day is dou- binding protein (RBP) were maintained on a vitamin ble-plotted. (a) Wild-type. (b) Cry17/7.(c) Cry27/7.(d) Cry17/7Cry27/7 A-deficient diet, ocular retinal was completely depleted, resulting in a mouse model in which all opsins, including classical opsins and novel opsins such as melanopsin, were rendered nonfunctional. We found circadian clock regulation (Miyamoto and Sancar, that these mice with nonfunctional opsin still retained 1998). mCry2 is expressed only weakly in the SCN normal gene induction by light, suggesting that (Figure 5b). The differential expression of Cry1 and cryptochromes are sufficient for circadian photore- Cry2 in retina and SCN, as well as the lack of sponses (Thompson et al., 2001). conservation of their C-termini, suggest the two Genetic analysis of a cryptochrome mutant in mammalian cryptochromes may have distinct roles in Drosophila, cryb, supports a role for cryptochrome as different tissues, although these proteins may share a circadian photoreceptor in fruitflies. These mutants some redundancy. synchronize poorly to light – dark cycles and do not respond to brief light pulses (Stanewsky et al., 1998). Under constant light conditions, wild-type flies become Genetic analysis behaviorally arrhythmic, but cryb flies remain rhythmic Genetic analysis of mice with null alleles of crypto- (Emery et al., 2000). In addition, only after elimination chromes demonstrated that cryptochromes have a role of both dCry and all known opsin photoreceptors does both in photoreception and in the circadian clock the fruitfly become circadian blind (Helfrich-Fo¨ rster et mechanism itself. Our behavioral analysis of locomotor al., 2001), suggesting that opsins and cryptochromes activity of individual cryptochrome knockout mice play redundant roles in fruitflies as they do in showed that cry17/7 mice have a shortened circadian mammals. period (22.5 h) (Figure 6b), while cry27/7 mice have a lengthened period (24.5 h) (Figure 6c) compared to Interactions with circadian clock proteins wild-type mice (23.7 h) (Figure 6a) (Thresher et al., 1998; Vitaterna et al., 1999; van der Horst et al., 1999), Cryptochromes have promiscuous interactions with suggesting cry1 and cry2 may play antagonistic roles in most circadian clock proteins. Cry1 and Cry2 exhibit regulating clock function. Behavioral studies of double light-independent interactions to different extents with cryptochrome knockout mice showed that their activity PP5, Bmal, Clock, Per1, Per2, and Per3, as shown by was still coordinated with light – dark cycles, but in the yeast two-hybrid assay (Zhao and Sancar, 1997;

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9052

Figure 7 Acute fos gene induction by light in the SCN. Data for four different mouse genotypes are shown: wild-type, rd (retinal degeneration), cry17/7cry27/7, and rd cry17/7cry27/7. The upper panel, Representative slices of peak fos signal in the SCN after each light dose. The lower panel, dose-response plot of c-fos induction in the SCN. Bars indicate standard deviation

Griffin et al., 1999; Shearman et al., 2000) and Shearman et al., 2000). Timing of nuclear entry of coimmunoprecipitation (Kume et al., 1999), and these these regulatory proteins is critical to proper main- interactions are presumed to govern a transcriptional tenance of circadian rhythmicity, and several feedback loop. The promoters of many circadian- mechanisms controlling nuclear entry have been regulated genes, including per genes, cry genes, and reported. Dimerization of Cryptochromes and Period output genes such as vasopressin, contain E-box proteins promotes nuclear entry of Per proteins (Kume elements which control circadian regulation of tran- et al., 1999), although in cry17/7cry27/7 knockout scription by a Clock – Bmal complex. Co-transfection mouse fibroblasts, nuclear entry of Per is unaffected assays for transcriptional activation of a luciferase (Yagita et al., 2000). In addition, casein kinase I (CKI) reporter from mPer1 E-box sequences demonstrated e and d interact with and phosphorylate Per proteins, that Cry strongly inhibits and Per weakly inhibits resulting in their cytoplasmic retention and degradation transcription induction by Bmal – Clock through direct (Vielhaber et al., 2000; Camacho et al., 2001) through a interaction (Griffin et al., 1999; Kume et al., 1999; ubiquitin-proteasome pathway (Akashi et al., 2002).

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9053

Figure 8 Circadian rhythm is regulated by a transcriptional feedback loop. Two different promoters exhibit different regulatory control loops. E-box promoters are activated by Bmal and Clock, and Per and Cry exert negative regulation of their own promoters. The bmal promoter is negatively autoregulated, but is upregulated by Cry

However, Cry can bind to the Per – CKI complex In Drosophila the feedback loop is similar. dCLOCK through its interaction with Per and translocate the and dCYCLE (dBMAL) activate transcription through complex to the nucleus, and in this complex Cry the E-box in the promoteres of per and tim. dPER and becomes a substrate for CKI (Eide et al., 2002), but the dTIM heterodimerize to enter the nucleus and down- physiological signiﬁcance of this phosphorylation is regulate E-box-mediated transcription. However, unknown. Regulation of the bmal promoter is although the transcriptional feedback loop in mammals controlled by a mechanism separate from E-box- is not directly light-sensitive, in ﬂies the interaction of mediated transcription. Cry upregulates bmal transcrip- dCRY with dPER and dTIM is light-dependent tion while Bmal exhibits negative autoregulation (Yu et (Rosato et al., 2001; Ceriani et al., 1999), and dCRY al., 2002). is degraded by light in a proteasome-dependent manner The current model for circadian clock regulation (Lin et al., 2001). consists of two interacting transcriptional feedback loops as shown in Figure 8. Transcriptional activators Bmal and Clock form a heterodimer that drives Conclusion transcription from E-box elements. Once per and cry genes are transcribed and translated, the resulting The photolyase/cryptochrome family of proteins regu- proteins dimerize in the cytoplasm, and re-enter the lates two distinct functions. Photolyase helps to nucleus to repress their own transcription by direct maintain the integrity of DNA after ultraviolet interaction with Bmal and Clock. Nuclear entry is damage, but does not exist in humans. The representa- controlled by phosphorylation of Per by CKI. In a tives of this family in humans are the cryptochromes, second auto-regulatory loop, Per and Cry positively which act as blue-light receptors for circadian entrain- regulate the bmal promoter, independent of the Bmal ment and as integral components of the molecular protein. While Bmal upregulates transcription from the clock mechanism. In mammals, there are multiple E-box, it downregulates its own transcription. The photoreceptors that contribute to circadian photore- mechanism of regulation of the bmal promoter is ception. Photoreceptors in the outer retina, the rod and unknown. cone opsins, mediate vision, but can also coordinate

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9054 absence of opsins, and in fact, appear to play the primary role in circadian photoreception (Figure 9), as evidenced by drastically reduced gene photoinduction in the SCN in Cry mutants and normal photoresponse in completely opsin-deficient mice. The existence of the circadian clock and its regulation by light has important implications for cancer etiology and treatment. Circadian oscillation of hormone levels may be involved in the development of hormone-sensitive cancers, such as breast cancer. Nocturnal light exposure results in acute suppression of plasma melatonin, a hormone that regulates sleep. However, melatonin also inhibits development of chemically-induced mammary tumors (Tamarkin et al., 1981), and light, which suppresses melatonin levels, increases the development of spontaneous mammary tumors in mice (Jo¨ chle, 1964; Anderson et al., 2000). In humans, a correlation was found between low night-time plasma melatonin levels and estrogen receptor positive breast cancer (Tamarkin et al., 1982). Further evidence for the role of circadian photoreception in breast cancer comes from the correlation between the incidence of blindness in women with breast cancer (0.15%) versus the incidence of bilateral blindness in a comparison group Figure 9 Schematic of circadian photoreception in mammals. (0.26%) (Hahn, 1991), and thus the ability to perceive The mammalian retina contains two classes of photoreceptors, light at night was found to correlate with incidence of opsins and cryptochromes. Cryptochromes in the inner retina ful- breast cancer. Circadian rhythms are also relevant to fill the primary role as circadian photoreceptor while classical opsins share some functional redundancy in regulating molecular the timing of cancer therapy regimens. With circadian and behavioral photoresponses variation in drug absorption, metabolism, and elimination, drug therapies can be optimized by coordinating drug delivery with the circadian rhythm light-regulated behavior in the absence of crypto- (Stevens and Rea, 2001) or perhaps eventually by chromes. Cryptochromes, located in the inner retina, control of circadian photoreception and manipulation are also sufficient to mediate photoresponses in the of the circadian clock.

References

Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Camacho F, Cilio M, Guo Y, Virshup DM, Patel K, Bult CJ, Lee NH, Kirkness EF, Weinstock KG, Gocayne Khorkova O, Styren S, Morse B, Yao Z and Geesler GA. JD and White O et al. (1995). Nature, 377, 3 – 174. (2001). FEBS Lett., 489, 159 – 165. Afonso CL, Tulman ER, Lu Z, Oma E, Kutish GF and Rock Ceriani MF, Darlington TK, Staknis D, Mas P, Petti AA, DL. (1999). J. Virol., 73, 533 – 552. Weitz CJ and Kay SA. (1999). Science, 285, 553 – 556. Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF and Rock Deisenhofer J. (2000). Mutat. Res., 460, 143 – 149. DL. (2000). J. Virol., 74, 3815 – 3831. Egan ES, Franklin TM, Hildebrand-Chae MJ, McNeil GP, Ahmad M and Cashmore AR. (1993). Nature, 366, 162 – 166. Roberts MA, Schroeder AJ, Zhang X and Jackson FR. Ahmad M, Jarillo JA, Smirnova O and Cashmore AR. (1999). J. Neurosci., 19, 3665 – 3673. (1998). Mol. Cell, 1, 939 – 948. Eide VJ, Vielhaber EL, Hinz WA and Virshup DM. (2002). J. Akashi M, Tsuchiya Y, Yoshino T and Nishida E. (2002). Biol. Chem., 277, 17248 – 17254. Mol. Cell. Biol., 22, 1693 – 1703. Eker AP, Dekker RH and Berends W. (1981). Photochem. Anderson LE, Morris JE, Sasser LB and Stevens RG. (2000). Photobiol., 33, 65 – 72. Cancer Lett., 148, 121 – 126. Eker APM, Hessels JKC and van de Velde J. (1988). Baer ME and Sancar GB. (1993). J. Biol. Chem., 268, 16717 – Biochemistry, 27, 1758 – 1765. 16724. Elich TD and Chory J. (1997). Plant Cell., 9, 2271 – 2280. Balsalobre A, Damiola F and Schibler U. (1998). Cell, 93, Emery P, Stanewsky R, Hall JC and Rosbash M. (2000). 929 – 937. Nature, 404, 456 – 457. Berson DM, Dunn FA and Takao M. (2002). Science, 95, Grifﬁn Jr EA, Staknis D and Weitz CJ. (1999). Science, 286, 1070 – 1073. 768 – 771. Briggs WR and Huala E. (1999). Annu. Rev. Cell. Dev. Biol., Guo H, Yang H, Mockler TC and Lin C. (1998). Science, 15, 33 – 62. 279, 1360 – 1363.

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9055 Hahn RA. (1991). Epidemiology, 9, 490 – 494. Park H, Kim S, Sancar A and Deisenhofer J. (1995). Science, Hattar S, Liao H, Takao M, Berson DM and Yau K. (2002). 268, 1866 – 1872. Science, 295, 1065 – 1070. Payne G, Heelis PF, Rohrs BR and Sancar A. (1987). Hays JB, Martin SJ and Bhatia K. (1985). J. Bacteriol., 161, Biochemistry, 26, 7121 – 7127. 602 – 608. Payne G and Sancar A. (1990). Biochemistry, 29, 7715 – 7727. Helfrich-Fo¨ rster C, Winter C, Hofbauer A, Hall JC and Petit C and Sancar A. (1999). Med. Sci., 12, 1411 – 1418. Stanewsky R. (2001). Neuron, 30, 249 – 261. Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF Hitomi K, Okamoto K, Daiyasu H, Miyashita H, Iwai S, and Rollag MD. (2000). J. Neurosci., 20, 600 – 605. Toh H, Ishiura M and Todo T. (2000). Nucleic Acids Res., Provencio I, Rollag MD and Castrucci AM. (2002). Nature, 28, 2353 – 2362. 415, 493. Hoffman PD, Batschauer A and Hays JB. (1996). Mol. Gen. Roenneberg T and Foster RG. (1997). Photochem. Photo- Genet., 253, 259 – 265. biol., 66, 549 – 561. Hsu DS, Zhao X, Zhao S, Kazantsev A, Wang R, Todo T, Rosato E, Codd V, Mazzotta G, Piccin A, Zordan M, Costa Wei Y and Sancar A. (1996). Biochemistry, 35, 13871 – R and Kyriacou CP. (2001). Curr. Biol., 11, 909 – 917. 13877. Sancar A. (1994). Biochemistry, 33, 2–7. Ishikawa T, Matsumoto A, Kato Jr T, Togashi S, Ryo H, Sancar A. (1996). Annu. Rev. Biochem., 65, 43 – 81. Ikenaga M, Todo T, Ueda R and Tanimura T. (1999). Sancar A. (2000). Annu. Rev. Biochem., 69, 31 – 67. Genes Cells, 4, 57 – 65. Sancar A, Franklin KA and Sancar GB. (1984). Proc. Natl. Jo¨ chle W. (1964). Ann. NY Acad. Sci., 117, 88 – 104. Acad. Sci. USA, 81, 7397 – 7401. Johnson JL, Hamm-Alvarez S, Payne G, Sancar GB, Sancar A and Sancar GB. (1988). Annu. Rev. Biochem., 57, Rajagopalan KV and Sancar A. (1988). Proc. Natl. Acad. 29 – 67. Sci. USA, 85, 2046 – 2050. Sancar GB and Sancar A. (1987). Trends Biochem. Sci., 12, Jorns MS, Sancar GB and Sancar A. (1984). Biochemistry, 259 – 261. 23, 2673 – 2679. Sancar GB and Smith FW. (1989). Mol. Cell. Biol., 9, 4767 – KanaiS,KikunoR,TohH,RyoHandTodoT.(1997).J. 4776. Mol. Evol., 45, 535 – 548. Sancar GB, Smith FW and Heelis PF. (1987a). J. Biol. KatoT,TodoT,AyokiH,IshizakiK,MoritaT,MitraSand Chem., 262, 15457 – 15465. Ikenaga M. (1994). Nucleic Acids Res., 22, 4119 – 4124. Selby CP and Sancar A. (1999). Photochem. Photobiol., 69, Kiener A, Husain I, Sancar A and Walsh C. (1989). J. Biol. 105 – 107. Chem., 264, 13880 – 13887. Selby CP, Thompson C, Schmitz TM, Van Gelder RN and Kim ST, Malhotra K, Smith CA, Taylor JS and Sancar A. Sancar A. (2000). Proc. Natl. Acad. Sci. USA, 97, 14697 – (1994). J. Biol. Chem., 269, 8535 – 8540. 14702. Kobayashi Y, Ishikawa T, Hirayama J, Daiyasu H, Kanai S, Shalitin D, Yang H, Mockler TC, Maymon M, Guo H, TohH,FukudaI,TsujimuraT,TeradaN,KameiY,Yuba Whitelam GC and Lin C. (2002). Nature, 417, 763 – 767. S, Iwai S and Todo T. (2000). Genes Cells, 5, 725 – 738. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves Komori H, Masui R, Kuramitsu S, Yokoyama S, Shibata T, I, Zheng B, Kume K, Lee CC, van der Horst GTJ, Hastings Inoue Y and Miki K. (2001). Proc.Natl.Acad.Sci.USA, MH and Reppert SM. (2000). Science, 288, 1013 – 1019. 98, 13560 – 13565. Shigeyoshi Y, Taguchi K, Yamamoto S, Takekida S, Yan L, Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Tei H, Moriya T, Shibata S, Loros JJ, Dunlap JC and Jin X, Maywood ES, Hastings MH and Reppert SM. Okamura H. (1997). Cell, 91, 1043 – 1053. (1999). Cell, 98, 193 – 205. Somers DE, Devlin PF and Kay SA. (1998). Science, 282, Li YF, Kim S and Sancar A. (1993). Proc. Natl. Acad. Sci. 1488 – 1490. USA, 90, 4389 – 4393. Srinivasan V, Schnitzlein WM and Tripathy DN. (2001). J. Lin C, Ahmad M and Cashmore AR. (1996). Plant J., 10, Virol., 75, 1681 – 1688. 893 – 902. Stanewsky R, Kaneko M, Emery P, Berette B, Wager-Smith LinC,YangH,GuoH,MocklerT,ChenJandCashmore K, Kay SA, Rosbash M and Hall JC. (1998). Cell, 95, 681 – AR. (1998). Proc. Natl. Acad. Sci. USA, 95, 2686 – 2690. 692. Lin C, Robertson DE, Ahmad M, Raibekas AA, Jorns MS Stevens RG and Rea MS. (2001). Cancer Causes Control, 12, and Cashmore AR. (1995). Science, 269, 968 – 970. 279 – 287. Lin F, Song W, Meyer-Bernstein E, Naidoo N and Sehgal A. Tamada T, Kitadokoro K, Higuchi Y, Inaka K, Yasui A, de (2001). Mol. Cell. Biol., 21, 7287 – 7294. Ruiter PE, Eker APM and Miki K. (1997). Nat. Struct. Malhotra K, Kim ST and Sancar A. (1994). Biochemistry, 33, Biol., 4, 887 – 891. 8712 – 8718. Tamarkin L, Cohen M, Roselle D, Reichert C, Lippman M Malhotra K, Kim ST, Walsh C and Sancar A. (1992). J. Biol. and Chabner B. (1981). Cancer Res., 41, 4432 – 4436. Chem., 267, 15406 – 15411. Tamarkin L, Danforth D and Lichter A. (1982). Science, 216, Malhotra K, Kim S, Batschauer A, Dawut L and Sancar A. 1003 – 1005. (1995). Biochemistry, 34, 6892 – 6899. Thompson CL, Blaner WS, Van Gelder RN, Lai K, Quadro Ma´ s P, Devlin PF, Panda S and Kay SA. (2000). Nature, 408, L, Colantuoni V, Gottesman ME and Sancar A. (2001). 207 – 211. Proc. Natl. Acad. Sci. USA, 98, 11708 – 11713. Miyamoto Y and Sancar A. (1998). Proc. Natl. Acad. Sci. Thresher RJ, Vitaterna MH, Miyamoto Y, Kazantsev A, USA, 95, 6097 – 6102. Hsu DS, Petit C, Selby CP, Dawut L, Smithies O, Miyamoto Y and Sancar A. (1999). Mol. Brain Res., 71, Takahashi JS and Sancar A. (1998). Science, 282, 1490 – 238 – 243. 1494. Ng WO and Pakrasi HB. (2001). Mol. Gen. Genet., 264, 924 – Todo T. (1999). Mutat. Res., 434, 89 – 97. 930. Todo T, Ryo H, Yamamoto K, Toh H, Inui T, Ayaki H, O¨ zerZ,ReardonJT,HsuDS,MalhotraKandSancarA. Nomura T and Ikenaga M. (1996). Science, 272, 109 – 112. (1995). Biochemistry, 34, 15886 – 15889.

Oncogene Blue light, DNA repair, and the circadian clock CL Thompson and A Sancar 9056 Todo T, Takemori H, Ryo H, Ihara M, Matsunaga T, Willer DO, McFadden G and Evans DH. (1999). Virology, Nikaido O, Sato K and Nomura T. (1993). Nature, 361, 264, 319 – 343. 371 – 374. Wood RD. (1997). J. Biol. Chem., 272, 23465 – 23468. TodoT,TsujiH,OtoshiE,HitomiK,KimSTandIkenaga Yagita K, Yamaguchi S, Tamanini F, van der Horst GTJ, M. (1997). Mutat. Res., 384, 195 – 204. Hoeijmakers JHJ, Yasui A, Loros JJ, Dunlap JC and van der Horst GTJ, Muijtjens M, Kobayashi K, Takano R, Okamura H. (2000). Genes Dev., 14, 1353 – 1363. Kanno S, Takao M, Wit J, Verkerk A, Eker APM, van Yang H, Wu Y, Tang R, Liu D, Liu Y and Cashmore AR. Leenen D, Buijs R, Bootsma D, Hoeijmakers JHJ and (2000). Cell, 103, 815 – 827. Yasui A. (1999). Nature, 398, 627 – 630. YasuiA,EkerAPM,YasuhiraS,YajimaH,KobayashiT, Vielhaber E, Eide E, Rivers A, Gao Z and Virshup DM. Takao M and Oikawa A. (1994). EMBO J., 13, 6143 – (2000). Mol. Cell. Biol., 20, 4888 – 4899. 6151. Vielhaber EL, Duricka D, Ullman KS and Virshup DM. Yu W, Nomura M and Ikeda M. (2002). Biochem. Biophys. (2001). J. Biol. Chem., 276, 45921 – 45927. Res. Commun., 1290, 933 – 941. Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, Zhao S and Sancar A. (1997). Photochem. Photobiol., 66, FruechteEM,HitomiK,ThresherRJ,IshikawaT, 727 – 731. Miyazaki J, Takahashi JS and Sancar A. (1999). Proc. Zhao X, Liu J, Hsu DS, Zhao S, Taylor JS and Sancar A. Natl. Acad. Sci. USA, 96, 12114 – 12119. (1997). J. Biol. Chem., 272, 32580 – 32590.

Oncogene