Structure and Function of Photolyase and in Vivo Enzymology: 50th Anniversary

Photoreactivation is the reversal of the harmful effects of far-UV radiation (200–300 nm) on organisms, such as growth delay, mutation, cell death, and cancer, by concomitant or subsequent exposure of the organism to near-UV/blue light (300–500 nm). The two major lesions induced in DNA by UV light are cyclobutane pyrimidine dimers (Pyr<>Pyr 2The abbreviations used are: Pyr, pyrimidine; CPD, cyclobutane pyrimidine dimer; MTHF, 5,10-methenyltetrahydrofolate; 8-HDF, 8-hydroxy-5-deazariboflavin; FRET, fluorescence resonance energy transfer. or CPD), which constitute ∼80–90% of the photoproducts, and pyrimidine-pyrimidone (6-4) photoproducts (Pyr[6-4]Pyr), which account for the 10–20% of the UV lesions. Photoreactivation results from the repair of these lesions in situ by flavoproteins called photoreactivating enzymes (photolyase) that use a blue-light photon as a co-substrate. Photolyases that repair these two photoproducts are evolutionarily related but functionally distinct. Enzymes that repair CPDs are referred to as CPD photolyase, and enzymes that repair (6-4) photoproducts are called (6-4) photolyase. For historical reasons and as a matter of common practice, the term “photolyase” without further qualification means CPD photolyase, and it will be used as such in this review, which celebrates the 50th anniversary of the discovery of photolyase. Photoreactivation was discovered by Kelner, who found that the lethal effect of UV radiation on Streptomyces griseus could be reversed if, following UV radiation, the irradiated bacterial culture was exposed to visible light (1Kelner A. Proc. Natl. Acad. Sci. U. S. A. 1949; 35: 73-79Crossref PubMed Google Scholar). Resurrection of the UV light-killed cells by light attracted the interest of many physicists in part because the reversal of the effect of high energy UV light by lower energy blue light was counterintuitive and seemed to run counter to the laws of physics. One such physicist was Claud S. Rupert, who eventually discovered photolyase (2Rupert C.S. Goodgal S.H. Herriott R.M. J. Gen. Physiol. 1958; 41: 451-471Crossref PubMed Scopus (96) Google Scholar). He and his colleagues used the DNA transformation assay to understand the molecular basis of photoreactivation. Extensive screening had revealed that photoreactivation was not universally distributed in the biological world. Of note, it was known that Escherichia coli possessed photoreactivation, but Haemophilus influenzae, which is a naturally transformable species, did not. Rupert did the following experiment (Fig. 1). He irradiated DNA isolated from a streptomycin-resistant H. influenzae strain with a UV dose that reduced the transformation efficiency by 30-fold. Then, he mixed the irradiated DNA with cell-free extract made from either H. influenzae or E. coli, and the mixtures were incubated either in dark or under light and used to transform a streptomycin-sensitive H. influenzae strain. He found that incubating the damaged DNA with the H. influenzae extract either in dark or under light did not improve its transformation efficiency. In contrast, whereas incubating the damaged DNA with E. coli extract in the dark did not affect its transforming capacity, light increased its transforming efficiency by 10-fold. Rupert and colleagues concluded that E. coli contained a light-activated enzyme that repaired the UV light-induced DNA damage and named it photoreactivating enzyme (2Rupert C.S. Goodgal S.H. Herriott R.M. J. Gen. Physiol. 1958; 41: 451-471Crossref PubMed Scopus (96) Google Scholar), which later came to be known as photolyase. Rupert continued to study the repair reaction in some detail using cell-free extracts or partially purified enzyme from E. coli and budding yeast and developed Scheme 1. E+UV⋅DNA⇄k2k1E⋅S→hvE⋅P⇄E+P SCHEME 1 Thus, he concluded that the reaction proceeds by the classical Michaelis-Menten scheme with the notable exception that catalysis is absolutely dependent on light. Finally, Rupert introduced the then nascent flash photolysis technology to the field of photolyase (Fig. 1) and using this technology determined some of the fundamental enzymatic parameters both in vitro and in vivo (3Harm W. Harm H. Rupert C.S. Mutat. Res. 1968; 6: 371-385Crossref PubMed Scopus (70) Google Scholar, 4Sancar A. Rupert C.S. Gene (Amst.). 1978; 4: 295-308Crossref PubMed Scopus (47) Google Scholar). Shortly after the discovery of photolyase, Pyr<>Pyr and (6-4) photoproducts were identified as the two major UV light-induced lesions in DNA. Photolyases from E. coli and budding yeast studied by Rupert and others repair only Pyr<>Pyr (5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). Recombinant DNA technology considerably accelerated the pace of characterization of these enzymes (4Sancar A. Rupert C.S. Gene (Amst.). 1978; 4: 295-308Crossref PubMed Scopus (47) Google Scholar, 5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar, 7Sancar A. Smith F.W. Sancar G.B. J. Biol. Chem. 1984; 259: 6028-6032Abstract Full Text PDF PubMed Google Scholar). However, it was only in 1993 that a photolyase that repairs the (6-4) photoproduct was also discovered (8Todo T. Takemori H. Ryo H. Ihara M. Matsunaga T. Nikaido O. Sato K. Nomura T. Nature. 1993; 361: 371-374Crossref PubMed Scopus (259) Google Scholar). Similarly, in 1993, it was found that an Arabidopsis thaliana protein with high sequence homology to photolyase had no repair activity but functioned as a blue-light receptor (cryptochrome) for plant growth and development (9Ahmad M. Cashmore A.R. Nature. 1993; 366: 162-166Crossref PubMed Scopus (1018) Google Scholar). Finally, in 1996, cryptochrome was discovered in humans (10Hsu D.S. Zhao X. Zhao S. Kazantsev A. Wang R.P. Todo T. Wei Y.F. Sancar A. Biochemistry. 1996; 35: 13871-13877Crossref PubMed Scopus (263) Google Scholar) and mice, and in 1998, it was shown to regulate the circadian clock in these and other animals by light-dependent and light-independent mechanisms (11Miyamoto A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6097-6102Crossref PubMed Scopus (372) Google Scholar, 12Thresher R.J. Vitaterna M.H. Miyamoto Y. Kazantsev A. Hsu D.S. Petit C. Selby C.P. Dawut L. Smithies O. Takahashi J.S. Sancar A. Science. 1998; 282: 1490-1494Crossref PubMed Scopus (333) Google Scholar, 13Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1988; 95: 681-692Abstract Full Text Full Text PDF Scopus (776) Google Scholar). In this review, E. coli photolyase, which is the best characterized photolyase to date, will be discussed, and when necessary, it will be compared with some other photolyases that have been studied in some detail. Then, the structures and functions of (6-4) photolyase and cryptochrome will be reviewed briefly before addressing the issue of suitability of photolyase for in vivo enzymology. Primary Structure—The photolyase/cryptochrome proteins, which are ∼400–600 amino acids in length, constitute a large and ancient flavoprotein family (14Partch C.L. Sancar A. Photochem. Photobiol. 2005; 81: 1291-1304Crossref PubMed Scopus (105) Google Scholar). Although sequence analysis is often sufficient to designate a new member of the family as a photolyase, a (6-4) photolyase, or a cryptochrome, frequently, the designation can be made only by functional testing. The three functional members of the family are not universally distributed. E. coli has only photolyase; marsupials have photolyase and cryptochrome; Drosophila possesses all three; placental mammals have only cryptochrome; and Bacillus subtilis and Caenorhabditis elegans have none. Cofactors and Chromophores—All photolyases are known or presumed to contain FAD as the catalytic cofactor (5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). In addition, they contain a “second chromophore” that is not essential for activity but increases the efficiency of repair under limiting light conditions (15Jorns M.S. Sancar G.B. Sancar A. Biochemistry. 1984; 23: 2673-2679Crossref PubMed Scopus (128) Google Scholar, 16Johnson J.L. Hamm-Alvarez S. Payne G. Sancar G.B. Rajagopalan K.V. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2046-2050Crossref PubMed Scopus (165) Google Scholar, 17Eker A.P.M. Hessels J.K.C. van de Velde J. Biochemistry. 1988; 27: 1758-1765Crossref Scopus (71) Google Scholar). The second chromophore, which functions as a light-harvesting photoantenna, is 5,10-methenyltetrahydrofolate (MTHF) in the majority of photolyases analyzed to date. In some rare species that can synthesize 5-deazaflavin, such as Anacystis nidulans, the second chromophore is 8-hydroxy-5-deazariboflavin (8-HDF). Finally, in some thermophilic bacteria, FMN and FAD have recently been identified as the second chromophores (18Ueda T. Kato A. Kuramitsu S. Terasawa H. Shimada I. J. Biol. Chem. 2005; 280: 36237-36243Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Fujihashi M. Numoto N. Kobayashi Y. Misushima A. Tsujimura M. Nakamura A. Kawarabayasi Y. Miki K. J. Mol. Biol. 2007; 365: 903-910Crossref PubMed Scopus (60) Google Scholar). The E. coli photolyase in its native state contains FAD in the two-electron reduced and deprotonated FADH– form (λmax = 360 nm, ϵ360 = 5,000 m–1 cm–1), and hence, the yellow color of the enzyme is dominated by MTHF (λmax = 385 nm, ϵ385 = 25,000 m–1 cm–1). However, during purification under aerobic conditions, FADH– is oxidized to the rather stable FADH· blue neutral radical, and the enzyme exhibits dark blue color. Excessive handling of the enzyme causes further oxidation to oxidized FAD, and the enzyme acquires a bright yellow color (5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). Crystal Structure of Photolyase—Crystal structures of several photolyases and of the cryptochrome from A. thaliana have been determined. All have the same basic architecture even when the sequence identity between members is as low as 25% and the enzymes have different second chromophores as in the case of E. coli photolyase, which contains MTHF, and A. nidulans photolyase, which contains 8-HDF (20Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (500) Google Scholar, 21Tamada T. Kitadokoro K. Higuchi Y. Inaka K. Yasui A. de Ruiter P.E. Eker A.P.M. Miki K. Nat. Struct. Biol. 1997; 4: 887-891Crossref PubMed Scopus (193) Google Scholar). Hence, as a representative of the entire family, the structure of the E. coli photolyase will be discussed (20Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (500) Google Scholar). The enzyme is essentially globular in shape (Fig. 2) and is made up of two well defined domains, an N-terminal α/β-domain (residues 1–131) and a C-terminal α-helical domain (residues 204–472). The two domains are to with a (residues that the α/β-domain (Fig. The MTHF is in a between the two In to MTHF, the FAD cofactor is the α-helical domain and is in by with amino The photolyase/cryptochrome FAD has the of the on of the is to be for the of to and the subsequent of the enzyme a that the of the In the of this a of the and to a Pyr<>Pyr to the FAD in the of the (Fig. that the is the to be repaired and then Finally, the structure revealed of the enzyme of functional have shown that the second chromophore to repair by fluorescence resonance energy to the catalytic FADH– (5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). to the efficiency of is to the between the and and to the between the of the and is when the is and the of the and have the same and The is in E. coli photolyase (20Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (500) Google Scholar) and in A. nidulans photolyase T. Kitadokoro K. Higuchi Y. Inaka K. Yasui A. de Ruiter P.E. Eker A.P.M. Miki K. Nat. Struct. Biol. 1997; 4: 887-891Crossref PubMed Scopus (193) Google Scholar). the between the chromophores in E. coli photolyase, the energy efficiency from MTHF to FADH– is only G. Sancar A. Biochemistry. PubMed Scopus Google Scholar) compared with the energy efficiency of in the A. nidulans enzyme S.T. Sancar A. Biochemistry. PubMed Scopus Google Scholar). The structures of these two enzymes an to this in E. coli photolyase, the of the and are with (20Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (500) Google Scholar). In contrast, the between the of the two chromophores in A. nidulans photolyase is only T. Kitadokoro K. Higuchi Y. Inaka K. Yasui A. de Ruiter P.E. Eker A.P.M. Miki K. Nat. Struct. Biol. 1997; 4: 887-891Crossref PubMed Scopus (193) Google Scholar), sufficient to for the and efficiency. catalysis to Michaelis-Menten and the the enzyme Pyr<>Pyr of light to form an which then a photon to Although the is common in photolyase from all other such enzymes in that the second is a photon and not a DNA and have shown that a the by the major and it by (5Sancar A. Biochemistry. 1994; 33: 2-9Crossref PubMed Scopus (569) Google Scholar, 6Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). of the DNA are by photolyase, which a stable between the on the of the enzyme and the and the three to the on the damaged and some with the of the from the I. Sancar G.B. Sancar A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). further the in the of the to of the the in the of the DNA (20Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (500) Google Scholar) to a high this the van with FADH– such that high efficiency can during is the case with other enzymes that in the of photolyase to a DNA with to a because it is to a from DNA from a A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1032) Google Scholar). The structure of the of A. nidulans photolyase with a a has the and has a of the A. T. P. U. Eker T. Science. PubMed Scopus Google Scholar). In to and photolyase and repairs In even a photolyase with and is repaired S.T. Sancar A. Biochemistry. PubMed Scopus Google Scholar). is that a of photolyases with to Pyr<>Pyr in DNA was recently discovered C.P. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 103: PubMed Scopus Google Scholar, Y. R. Smith C.L. C.L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. 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Res. 1968; 6: 371-385Crossref PubMed Scopus (70) Google Scholar). was because of three the of catalysis by photolyase on light it to the and catalysis the of in the cell can be by the UV dose to the cell Finally, a light flash of is sufficient to all of the to the enzyme in a in flash have been used to the of the photolyase the reaction a flash is sufficient for flash photolysis was used in vivo to some basic parameters such as the of photolyase in the the and and the of the and the of the and the of the of repair (3Harm W. Harm H. Rupert C.S. Mutat. Res. 1968; 6: 371-385Crossref PubMed Scopus (70) Google Scholar). when the E. coli photolyase was eventually purified and the with the defined I. Sancar A. Res. Scopus (71) Google Scholar, Sancar A. Biochemistry. PubMed Scopus Google Scholar) to in vivo 1). the the and other and of photolyase could not be by the in vivo flash photolysis photolyase a in because of its to in vivo enzymology. 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The light of catalysis has made it to the of the reaction reaction in The same of the enzyme has in vivo which is a major of in the Finally, the characterization of photolyase a in the discovery of cryptochrome in and the discovery of the related but functionally in have to blue-light in the circadian clock and in and in have that an experiment with H. influenzae DNA and E. coli cell-free extract have major on from in to in and to in and