Genes Genet. Syst. (2007) 82, p. 311–319 Biochemical and biological properties of DNA derived from utraviolet-sensitive rice cultivars

Ayumi Yamamoto1, Tokuhisa Hirouchi1, Tamiki Mori1, Mika Teranishi1, Jun Hidema1, Hiroshi Morioka2, Tadashi Kumagai1 and Kazuo Yamamoto1*† 1Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan 2Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan

(Received 5 June 2007, accepted 25 July 2007)

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

Key words: chromophore, cyclobutane (CPD), DNA photolyase, Oryza sativa,

class II photolyases are found in some , archea, INTRODUCTION , green algae, insects, and vertebrates (Yasui et al., Ultraviolet light (UV) causes DNA damage such as the 1994; O’Connor et al., 1996; Ahmad et al., 1997; Petersen cyclobutane pyrimidine dimer (CPD) and pyrimidine et al., 1999; Kim et al., 1996; Kato et al., 1994). Class I pyrimidone 6-4 photoproduct which are known to kill cells photolyases have generally two kinds of chromophore; by blocking DNA replication and transcription (Friedberg reduced flavin-adenine dinucleotide (FAD) as a catalytic et al., 1995), and if damaged DNA is replicated this might and either 8-hydroxy-5-deazaflavin or 5,10-meth- lead to mutagenic events (Hutchinson et al., 1988; Tanaka enyl tetrahydrofolate (MTHF) as a light harvesting factor et al., 2001). CPD is a substrate for the UV-A/blue light- (Sancar, 2003). To date, the crystal structure of class I dependent repair activity of CPD photolyase (Dulbecco, photolyases from Escherichia coli, Anacystis nidulans and 1949). CPD photolyases are classified into two classes, I Thermus thermophilus has been solved without DNA and II, based on amino acid sequence similarity (Yasui et (Park et al., 1995;Tamada et al., 1997; Komori et al., al., 1994; Kanai et al., 1997; Todo, 1999). Class I photol- 2001) and that of A. nidurans has been solved with DNA yases are mostly found in bacteria and archea, whereas (Mees et al., 2004). X-ray analyses have revealed that the class I enzymes share a similar global fold, which Edited by Hirokazu Inoue consists of an amino-terminal α/β domain and a carboxy- * Corresponding author. E-mail: [email protected] † Present address: Department of Biomolecular Sciences, Gradu- terminal helical domain. The helical domain is composed ate School of Life Sciences, Tohoku University, Sendai 980- of clusters I and II, between which a cavity is formed where 8578, Japan the FAD is buried. X-ray analyses have also revealed the 312 A. YAMAMOTO et al. structures of CPD-photolyase complexes with base is the residue crucial for substrate-binding and repair. flipping at the cavity (Mees et al., 2004). The crystal Although we observed no difference in biochemical char- structure further indicated that the α/β domain is a site acter between Sasanishiki and Norin 1 GST-photolyases where a second chromophore can bind (Park et al., 1995; purified from E. coli, there were differences in UV sensi- Henry et al., 2004). tivity in vivo and differences in the biochemical activity In contrast to the class I photolyases, little is known of the photolyase preparations from Sasanishiki and about the structure of the class II CPD photolyases. Norin 1 cultivars (Hidema et al., 2000). At position 126, class II CPD photolyases purified from E. coli con- the codon CAG in Sasanishiki encodes glutamine and the tain the catalytic factor FAD but a second chromophore codon CGG in Norin 1 encodes arginine. We thus argue has not been detected (Kleiner et al., 1999). The second- that position 126 may be the site of binding of a second ary structure predicted from the amino acid sequence of chromophore, which has not yet been identified in rice. class II photolyases characterized to date consists of an amino-terminal α/β domain and a carboxy-terminal heli- MATERIALS AND METHODS cal domain (Slamovits and Keeling, 2004), but is less sim- ilar than the class I photolyase from E. coli. However, no Bacterial strains, plasmids and media The E. coli crystallographic or NMR information on class II photol- strains NKJ3002 (phr– uvrA– recA–) (Nakajima et al., yases or CPD-class II photolyase complexes is presently 1998), KY20 (phr–) (Hirouchi et al., 2003) and DH5α (phr+ available. recA–) (Grant et al., 1990) were used as hosts for the We have recently characterized a class II CPD photol- cloning of rice photolyase genes, overexpression and yase from Oryza sativa (Hidema et al. ,2000; Hirouchi et purification of GST-photolyase fusion proteins, and com- al., 2003; Teranishi et al., 2004). It has been indicated plementation of the rice photolyase gene in vivo. The that Asian rice cultivars differ in their response to UV plasmid pGEX-4T used for the glutathione-S- radiation in terms of growth and physiological processes (GST)-fused constructs was purchased from Amersham (Teramura et al., 1991). Actually, we observed that a Biosciences. The plasmid pKY1 carrys E. coli phr gene Japanese rice cultivar, Sasanishiki, a leading variety in (Yamamoto et al., 1983). northern Japan, exhibits more resistance to UV, whereas Luria (L) broth, L agar, and 0.067 M phosphate buffer Norin 1, a progenitor of many Japanese commercial rice were prepared as described previously (Akasaka and cultivars, is less resistant, although these cultivars are Yamamoto, 1991). Ampicillin (50 μg/ml) was included if closely related in terms of breeding (Teranishi et al., necessary in the L broth and L agar. 2004). We further found that the UV-sensitive Norin 1 is defective in CPD photoreactivation in vivo and in vitro Rice CPD photolyase genes CPD photolyase cDNAs (Hidema et al., 2000; Teranishi et al., 2004). A similar from Sasanishiki (Hirouchi et al., 2003), Norin 1 (Teranishi correlation between sensitivity to UV and a defective CPD et al., 2004) and Surjamkhi (Hidema et al., 2005) have photoreactivation was also observed in an UV-sensitive been described. A cDNA library of Gulfmont (Life cultivar of Surjamkhi, the indica rice cultivar (Hidema et Technology) was screened using oligonucleotide primers al. 2001; Hidema et al., 2005). Clarifying the molecular used for cloning the Sasanishiki photolyase gene, yielding nature of the deficiency of CPD photolyase in Norin 1, a Gulfmont CPD photolyase gene with a 1446-bp ORF cod- Surjamkhi, and other UV-sensitive rice cultivars, may ing for 481 amino acid residues (DDBJ/EMBL/GenBank help us to understand the structure and function of class databases under accession No. AB210109). To construct II CPD photolyases. a chimeric plasmid, full-length cDNA from Sasanishiki To understand the molecular nature of the differences and that from Surjamkhi were digested with XhoI and the in sensitivity to UV among rice cultivars derived from 5’-fragment from the Sasanishiki cDNA and 3’-fragment Sasanishiki, Norin 1, and Surjamkhi, we have constructed from the Surjamkhi cDNA were ligated to obtain cDNA GST-photolyase plasmids, purified enzymes from E. coli, named Sasa-jamkhi. and characterized the photolyases in vitro. We have also cloned photolyase cDNA from Gulfmont, which we have Photoreactivation and UV-damage binding effects of observed as one of the most UV sensitive japonica rice rice CPD photolyases in E. coli NKJ3002 (phr– uvrA– cultiver (unpublished result). Our biochemical results recA–) and DH5α (phr+ recA–) cells were transformed with indicate that photolyases from Sasanishiki and Norin 1 pGST-photolyase plasmids, and the transformants were have similarly high levels of repair activity, whereas pho- grown to log phase in L broth containing ampicillin. tolyases from Surjamkhi and Gulfmont are weak or defec- Samples of 5.0 ml were irradiated in 90-mm diameter tive, respectively, in substrate binding and photorepair. petri dishes with UV light (254 nm) provided by a germi- The difference is attributed to the replacement of an cidal lamp. The fluence rate was 0.025 J/m2/s for amino acid at position 296, glutamine in Sasanishiki and NKJ3002 and 0.35 J/m2/s for DH5α. DH5α samples were Norin 1 with histidine in Surjamkhi and Gulfmont, which plated on L agar and incubated at 37°C overnight to score Characterization of photolyase from rice 313 surviving colonies. NKJ3002 samples were illuminated Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, and 1 mM with a fluorescent lamp for 30 min as described previ- DTT in the dark at 25°C for 15 min and the mixture was ously (Yamamoto et al., 1985), plated on L agar and incu- electrophoresed on a non-denatured acrylamide gel as bated at 37°C overnight. described previously under yellow light (Nakajima et al., 1998). The levels of band shifting were determined from Purification of the GST-CPD photolyase fusion pro- the intensity of the radiolabeled oligonucleotides using a teins The rice CPD photolyase cDNAs were amplified BASS 2000 imaging analyzer (Fuji Film). by PCR using two primers that annealed near the start and stop codons with new BamHI and EcoRI restriction Spectroscopic analysis The absorption spectra were sites at the 5’-end and 3’-end, respectively. The PCR recorded with a ND-1000 spectrophotometer (NanoDrop products were digested with BamHI and EcoRI and Technologies). The concentration of the apoprotein was inserted into the BamHI/EcoRI sites of pGEX-4T to con- determined with the Bradford method. To determine the struct the plasmids pGST-Sasanishiki, pGST-Norin 1, flavin concentration, the GST-Rice photolyase holopro- pGST-Surjamkhi, pGST-Gulfmont and pGST-Sasa-jamkhi, teins were heated at 95°C for 5 min in buffer containing which express the GST-fused form. 50 mM Tris-HCl, pH 8.0, and the precipitated protein was GST-fused photolyases were purified basically accord- removed by centrifugation. The absorption spectrum in ing to a protocol described previously (Hirouchi et al., the 250–600 nm range was recorded, and the flavin con- 2003). Briefly, E. coli KY20 (phr) cells transformed with centration was calculated from the absorbance at 450 nm pGST-plasmids were grown in L broth containing ampi- using a molar extinction coefficient of 11,300 M–1 cm–1. cillin at 25°C with 0.3 mM of isopropyl-β-D-thiogalacto- side for 10 h then collected by centrifugation. Cells were RESULTS harvested, resuspended in phosphate buffer, and sonicated. The debris was removed by centrifugation. The cell- Cloning of CPD photolyase genes from UV-sensitive free extract was loaded onto a column of glutathione- rice cultivars for characterization Rice Sepharose 4B (Amersham Pharmacia Biotech) and eluted plants were grown for 45 days under irradiation with with an elution buffer containing reduced glutathione. visible light supplemented with or without UV in a Fractions containing photolyase were combined and con- phytotron (Teranishi et al. 2004). Figure 1 shows the centrated to 100 μl using a centrifuge filter unit (Millipore variation in sensitivity to UV among the rice cultivars of Co., Bedford, MA). All operations were carried out at 4°C or on ice. A portion of the protein sample was sub- jected to SDS-PAGE, and the purity and concentration were estimated with an Image-analyzer and the Bradford method, respectively.

DNA Substrate Oligonucletotides (30 mers) containing a centrally located thymine dimer were prepared as pre- viously described (Iwai et al., 1994). The sequence of the substrate is 5’-CACGTACGCATCTTCTACGTACCGACA- GTC-3’ (the two T residues that make up the cyclobutane thymine dimer are underlined). Thirty pmoles of oligo- nucleotide containing the thymine dimer was 5’-end- labeled with [γ-32P]ATP and T4 polynucleotide kinase (TaKaRa), then annealed with 50 pmol of complementary oligomer in annealing buffer (10 mM Tris-HCl, pH 8.3, and 10 mM MgCl2) by heating at 95°C for 3 min and cool- ing to 30°C over a 30-min period. The duplex DNA was ethanol precipitated and resuspended in deionized dis- tilled water.

Band shift assay in vitro Thirty-mer DNA substrates which have a single thymine dimer were used in the band shift assay and the in vitro photoreactivation assay. For Fig. 1. Effects of UV radiation on rice cultivars Sasanishiki (A), μ Norin 1(B), Surjamkhi (C), and Gulfmont (D). Rice cultivars were the band shift assay, 0 to 3.0 g of partially-purified GST- grown for 45 days in a phytotron under visible radiation (350 32 fused protein was added to 1.5 nmol of the P-labeled μmol photon/m2/s) without (–UV) or with (+UV) supplemented UV DNA substrates in a reaction buffer containing 50 mM radiation from UV-B fluorescent bulbs (Teranishi et al., 2004). 314 A. YAMAMOTO et al.

Sasanishiki, Norin 1, Surjamkhi and Gulfmont. the cDNA was inserted into the glutathione-S-transferase Sasanishiki is the most resistant to UV radiation followed (GST) fusion vector and expressed in E. coli strain by Norin 1, Surjamkhi and Gulfmont in this order. To NKJ3002 (phr– uvrA– recA–). An efficient photoreactiva- understand the molecular nature of the difference in UV tion was observed in NKJ3002 carrying pGST-Sasanishiki sensitivity, we cloned the CPD photolyase genes from and pGST-Norin 1 (Fig. 3). On the other hand, NKJ3002 Sasanishiki (Hirouchi et al., 2003), Norin 1 (Teranishi et carrying pGST-Surjamkhi and pGST-Sasa-jamkhi showed al., 2004), Surjamkhi (Hidema et al., 2005), and Gulfmont weak photoreactivation as compared to NKJ3002 carry- (this study) as described in Materials and Methods. Fig- ing pGST-Sasanishiki (Fig. 3). The weakest photoreacti- ure 2 shows the alignments of amino acids derived from vation was observed in NKJ3002 carrying pGST- 4 rice caltivars, in which the Sasanishiki, Norin 1, and Gulfmont (Fig. 3). We next transferred pGST-photolyase Surjamkhi CPD photolyase genes encode a 1521-bp ORF plasmids into an excision-repair proficient E. coli phr+ coding for 506 amino acid residues, and the Gulfmont recA– strain (DH5α). In this case, while the photolyase CPD photolyase gene encodes a 1446-bp ORF coding for derived from E. coli can bind to CPD in the dark and 481 amino acid residues. enhance nucleotide excision repair, thus rendering the host cells more resistant to UV (Yamamoto et al., 1983), the photolyase derived from foreign species can bind to CPD and inhibit the access of E. coli photolyase and nucleotide excision repair, thus rendering the host cells more sensitive to UV (Kobayashi et al., 1990). As shown in Fig. 3B, DH5α cells carrying pGST-Sasanishiki and pGST-Norin 1 were the most sensitive to UV, indicating that both Sasanishiki photolyase and Norin 1 photolyase can efficiently bind CPD and inhibit the access of E. coli photolyase and nucleotide excision repair in the dark. Fig. 2. Alignment of amino acid sequences derived from UV- The DH5α cells carrying pGST-Surjamkhi and pGST- sensitive rice species. Sasa-jumkhi were moderately sensitive and those carry- ing pGST-Gulfmont were the most resistant to UV radia- At position 126, the codon CAG in Sasanishiki encodes tion in the dark (Fig. 3B). Using Western blotting, we glutamine (Gln) and the codon CGG in other species also observed that the amounts of photolyases expressed encodes arginine (Arg). Thus a G→A or A→G transition in NKJ3002 were essentially the same among the 5 spe- occurred. At position 255, the codon GGC in Sasanish- cies (data not shown). Thus, the results in DH5α indi- iki, Norin 1, and Surjamkhi encodes glycin (Gly), and the cate that in the ability to bind CPD in E. coli, the order codon AGC in Gulfmont encodes serine (Ser) (G→A or is Sasanishiki = Norin 1 > Surjamki = Sasa-junmkhi > A→G transition). At 296, the codon CAG in Sasanishiki Gulfmont. The results in NKJ3002 indicate that in and Norin 1 encodes Gln, and the codon CAC in Sur- terms of CPD photorepair ability in E. coli, the order is jamkhi and Gulfmont encodes His (C→G or G→C trans- again Sasanishiki = Norin 1 > Surjamki = Sasa-junmkhi version). We further found that nucleotide A at position > Gulfmont. Concerning the effect of Gulfmont photol- 1424 in Gulfmont was lost leading to a mRNA one base yase, the difference to UV radiation between DH5α car- short encoding a truncated 481-amino acid protein. rying vacant vector and pGST-Gulfmont was very small Before purifying and characterizing the rice CPD pho- but reproducible, and the difference of photorepair ability tolyases, we constructed a chimeric CPD photolyase com- between NKJ3002 carrying vacant vector and pGST-Gulf- prising a 5’-fragment of Sasanishiki and 3’-fragment of mont was very small but reproducible. Thus, we are Surjamkhi using the unique XhoI site at nucleotide assuming that Gulfmont photolyase has weak but signif- 898. Consequently, we obtained a cDNA named Sasa- icant ability to bind and photorepair CPD in E. coli. jamkhi which has Gln at 126, Gly at 255, and His at 296 (Fig. 2). The biological and biochemical characteristrics In vitro thymine dimer-binding activity of rice CPD of Gln296His could be examined using Sasa-jamkhi. photolyases The recombinant proteins were purified Thus, genetic and biochemical analyses were necessary to from extracts of E. coli KY20 (phr–) by affinity chromatog- assign differences in the functions of photolyases from raphy on a GST-column yielding a single band of 90 kDa UV-sensitive rice cultivars. as revealed by SDS-PAGE (Fig. 4). The proteins were > 80% pure, and therefore appropriate for spectroscopic and Photoreactivation and binding of UV-induced dam- enzymatic analysis. Thymine dimer-binding activity age in E. coli expressing the rice CPD photolyases was then tested in vitro. As shown in Fig. 5, the purified To determine how efficiently rice CPD photolyases can rice CPD photolyases bound to the thymine dimer sub- complement the deficiency of photoreactivation in E. coli, strate in an enzyme concentration-dependent manner. Characterization of photolyase from rice 315

Fig. 3. Photoreactivation and binding of UV-induced damage in E. coli strain NKJ3002 (phr– uvrA– recA–) and DH5α (recA–), respectively, expressing the rice CPD photolyases. Complementation assay (A); After UV irradiation at 254 nm, the NKJ3002 cells carrying the GST-photolyase plasmid were kept in the dark (closed square) or illuminated for 30 min with a day-light fluorescent bulb (open triangle). Plasmids used were pKY1 (E. coli photolyase gene), pGST-Sasanishiki, pGST-Norin 1, pGST- Surjamkhi, pGST-Sasa-jamkhi and pGST-Gulfmont. UV-damage binding (B); DH5α cells carrying rice photolyase plasmids were also irradiated with UV radiation and kept in the dark without illumination. Plasmids used were vacant vector (ve), pGST-Gulfmont (Gu), pGST-Surjamkhi (Su), pGST-Sasa-jamkhi (Sj), pGST-Norin 1 (No), and pGST-Sasanishiki (Sa).

tion of a photolyase-thymine dimer complex was deter- mined by incubating increased amounts of enzyme (for Sasanishiki, Norin 1, Surjamkhi and Sasa-jamkhi, 0 to 350 ng, and for Gulfmont, 0 to 850 ng) with a constant amount of substrate (150 fmol) and quantitating the frac- tion of enzyme-bound DNA at each concentration. From

the Schatchard plots shown in Fig. 6, we obtained a Ka value of 1.4 × 107 M–1 for Sasanishiki photolyase, 1.1 × 107 M–1 for Norin 1 photolyase, 1.8 × 106 M–1 for Surjamkhi photolyase, 1.1 × 106 M–1 for Sasa-jumkhi photolyase, and 3.7 × 105 M–1 for Gulfmont photolyase. The values for Sasanishiki and Norin 1 are in the similar range as the Fig. 4. Coomassie blue staining of GST-rice photolyases. GST- 8 –1 Ka of E. coli CPD photolyase (2.6 × 10 M ) (Sancar, rice photolyases were eluted on glutathione-Sepharose, and sep- 1994). Compared with the values for the Sasanishiki and arated using a 4–20% gradient. Lane 1 contains marker pro- teins, lane 2 Sasanishiki, lane 3 Norin 1, lane 4 Surjamkhi, lane Norin 1 enzymes, the Ka for the Surjumkhi and Sasa- 5 Gulfmont and lane 6 Sasa-jamkhi. An arrow indicates the 90- jumkhi (Gln296His) photolyases was decreased by one kDa protein band. order of magnitude whereas the Ka for the Gulfmont (Gly255Ser, Gln296His and N-terminal truncated) The photolyases from Sasanishiki and Norin 1 bound enzyme was decreased by two orders of magnitude. strongly to the 30-mer substrates in the dark. The pho- Thus, among the 5 photolyases, the Sasanishiki and tolyases from Surjamkhi and Sasa-jamkhi bound weakly Norin 1 photolyases are the most proficient at binding the to the 30-mer substrates (Fig. 5). The photolyase from thymine dimer followed by the Surjamkhi and Sasa- Gulfmont barely bound the thymine dimer at all (Fig. jamkhi enzymes. The Gulfmont photolyase is the weak-

5). The equilibrium binding constant, Ka, for the forma- est of the 5 (Figs. 3B and 5B). 316 A. YAMAMOTO et al.

Fig. 5. Gel shift analysis showing thymine dimer-binding activity of GST-rice photolyases. 32P-labeled nondimer 30 mer (lane 1) or 30 mer with thymine dimer (lane 2 to 5) was mixed with 0 to 3.0 μg of GST-photolyases in a reaction buffer con- taining 50 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT in the dark at 25°C for 15 min and the mixture was electrophoresed on a non-denatured acrylamide gel (A). The bound and unbound fractions of 30-mer substrates in A were quantified with a bioimaging analyzer (B). A shifted band indicates the percentage of bound fraction per sum of the bound and unbound fractions. Each point in (B) represents an average of 3–4 independent experiments with standard error shown. The results of (A) represent one experiment done at same time.

Sasanishiki photolyase contains FAD (Hirouchi et al. 2003). Thus, we wanted to know whether the difference in photolyase activity among the 5 photolyases is due to differences in chromophore-binding. Figure 7 shows absorption spectra of GST-photolyases from Sasanishiki and Gulfmont. The absorption spectra from Norin 1, Surjamkhi, and Sasa-jamkhi were essentially the same as

Fig. 6. Analysis of the gel retardation data (Fig. 5) by Scatchard plots. In this experiment, 150 fmol of substrate and 0 to 350 ng of enzyme from Sasanishiki (filled diamond), Sasa-jamkhi (X), Norin 1 (filled square), and Surjamkhi (filled triangle) were used. In the case of Gulfmont (filled circle), 150 fmol of substrate and 0 to 850 ng of enzyme were mixed.

Fig. 7. Absorption spectra in the 300–550 nm range of the puri- Spectroscopic properties of rice photolyases All fied GST-Sasanishiki and Gulfmont enzymes. Spectroscopic anal- photolyases characterized to date contain FAD as a cata- yses of native (A) and boiled (B) GST-rice photolyases, compared lytic chromophore. We have previously shown that the with standard FAD (C). Characterization of photolyase from rice 317 those from Sasanishiki and Gulfmont (data not shown). the photolyase gene, causing alterations of three deduced The spectra derived from the Sasanishiki photolyase amino acids at positions 126, 255, and 296 (Fig. 2). We exhibited peaks in the near-UV/blue light region, at 360 further constructed a chimeric gene using the Sasanishiki nm, 450 nm and 475 nm, and a shoulder at 420 nm. To 5’ fragment and Surjamkhl 3’ fragment, named Sasa- confirm the differences in the amount of FAD in photol- jamkhi. Thus, Sasa-jamkhi has the same amino acid yases, the proteins were heat-denatured at neutral pH, composition as Sasanishiki at positions 126 and 259 and and, following removal of the denatured proteins by cen- the same composition as Sarjamkhi at position 296 (Fig. trifugation, the absorption spectrum was recorded. The 2). Using GST-constructs, we have purified enzymes absorption spectrum of the chromophore released from from E. coli. Among these GST-constructs, Sasanishiki the photolyase was typical of that of flavin with maxima and Norin 1 photolyases are almost equally effective at at 375 nm and 450 nm (Fig. 7, B). The stoichiometries complementing phr-negative E. coli, efficiently bind UV- of FAD relative to the apoprotein were roughly estimated damage in recA– E. coli in the dark, and bind the thymine from the ratio of the absorption of the denatured enzyme dimer in vitro in the dark (Figs. 3 and 5). Surjamkhi and at 450 nm to the amount of the holoenzyme determined Sasa-jamkhi photolyases can weakly complement phr- by the Bradford method; the apoprotein : FAD ratio in negative E. coli, bind UV-damage in recA– E. coli in the Sasanishiki photolyase was 1 : 0.5 and that in Gulfmont dark, and bind the thymine dimer in vitro in the dark was 1 : 0.12. We further calculated an apoprotein : FAD (Figs. 3 and 5). The Gulfmont photolyase is the weakest ratio of 1 : 0.48 in Norin 1, and 1 : 0.25 in Sasa-jumkhi. at complementing phr-negative E. coli, binding UV- Although information on FAD-binding in Surjamkhi is damage in recA– E. coli in the dark, and binding the not available, the difference in activities in complement- thymine dimer in vitro in the dark (Figs. 3 and 5). Thus, ing the phr-defect in E. coli in vivo, binding UV-damage among the three amino acids altered, His at position 296 in vivo in E. coli, and binding the thymine dimer in vitro in photolyases from Surjamkhi, Gulfmont and Sasa- among the 5 photolyases are directly correlated with the jamkhi but not Gln at position 296 in photolyases from number of FAD residues bound to the apoenzyme. It is Sasanishiki and Norin 1, is important for binding to and known that E. coli photolyase unbound to FAD cannot thus repairing CPD. bind CPD (Payne and Sancar, 1990). Concerning the amino acid sequence at and near posi- tion 296, conservation was seen not only in class II CPD photolyases but also in the E. coli CPD photolyase (class DISCUSSION I) (Fig. 8). In the E. coli CPD photolyase, these sites are Two classes of CPD photolyases are known, I and II. The native structure of class I CPD photolyases has been determined. The repair reaction of class I CPD photol- yases has been characterized as well. A Class I CPD photolyase binds CPD in DNA independent of light, and flips the dimer out of the double helix into the cavity to make a stable enzyme-substrate complex. Thus, it is assumed that the MTHF absorbs a near-UV/Blue- light photon and transfers the excitation energy to flavin, which then transfers an electron to CPD, generating a dimer radical anion. This anion is unstable and under- goes spontaneous splitting to the original undamaged two pyrimidines (Sancar, 2003). In contrast, the repair reac- tion of class II CPD photolyases is unclear. Moreover, both class I and class II CPD photolyases have the same catalytic chromophore, FAD, and bind the same sub- strate, pyrimidine dimers, implying that the structure Fig. 8. Sequence homology among 5 class II photolyases and one class I photolyase. The positions of the mutation in rice are and function of class II photolyases are similar to those indicated in bold face at 196, 255 and 296. + marks E. coli res- of class I CPD photolyases. idues known to interact with MTHF (Park et al., 1995; Henry et To clarify the repair mechanism of class II CPD photol- al., 2004), * marks FAD binding (Park et al., 1995; Mees et al., yases, we wanted to characterize CPD photolyases deri- 2004) and x mark CPD binding (Mees et al., 2004). Species ved from rice cultivars with different UV sensitivities in abbreviations are: Os, O. sativa Sasanishiki BAC76449; Ag, vivo; Sasanishiki is the most resistant followed by Norin Anopheles gambiae EAA09350; Ca, Carassius auratus D11391; At, Arabidopsid thaliana X99301; So, Spinacia oleracea AAP31407; 1, Surjamkhi and Gulfmont in this order (Fig. 1). The Ec, Eschercihia coli. Alignment was achieved with the Clustal W deficiency or proficiency in photoreactivation in these rice program available at http://searchlauncher.bcm.tmc.edu/multi- cultivars is attributed to three changes of nucleotide in align/multi-align.html and with eye. 318 A. YAMAMOTO et al. known to interact with the catalytic cofactor, FAD (* in second chromophore was detectable, differences in the Fig. 8; Park et al., 1995) and CPD (× in Fig 8; Mees et al., ability to bind MTHF between the Sasanishiki and Norin 2004). We therefore assumed that the alteration of 1 enzymes had no influence on the enzyme activity, lead- Gln296His in the Surjamkhi, Gulfmont, or Sasa-jamkhi ing to the same complementing, binding, and repairing photolyase might lead to a defect in binding to FAD. It activity. In class II CPD photolyases, no amino acid has been reported that an apoenzyme without FAD had sequence conservation at position 126 was seen, however, no affinity for CPD but regained the ability to bind CPD in the vicinity at 126, conservation was seen (Fig. 8). upon binding stoichiometrically to FAD (Payne and Rice is one of the world’s most important staple food Sancar, 1990). Our spectroscopic observation indicates grains and is widely cultivated throughout Asia. Rice is the stoichiometrical binding of FAD to the Sasanishiki grown not only in regions of moderate climate at middle and Norin 1 enzymes. On the other hand, the Sasa- latitudes but also in tropical climates, such as in Indone- jamkhl and Gulmont photolyases bound to FAD at a rate sia or the Bengal region, where the amount of UV radia- of 1 : 0.25, and 1 : 0.12, respectively. Thus, we argue tion in sunshine is greater. Thus, systematic breeding of that Gln at position 296 is involved in the binding of FAD rice cultivars tolerant of UV radiation is particularly not in the binding of thymine dimer. To date, using class helpful in tropical regions where there is more UV radia- I photolyases, there have been no direct reports suggest- tion in sunshine. As described before, there are strong ing position 296 as a FAD-. Analyzing the correlations between sensitivity to UV and growth retar- crystal structure of class II photolyase should determine dation or productivity in rice plants (Hidema et al., 2000; whether Gln296 is the site where FAD binds or not. Hidema et al., 2001; Hidema et al., 1997). Since photo- It was found previously that Norin 1 was sensitive to reactivation is the major pathway protecting against the UV radiation compared with Sasanishiki (Hidema et al., deleterious effects of UV radiation in plants (Britt, 1999), 2000; Hidema et al., 1997; Hidema and Kumagai, 1998; the introduction of the CPD photolyase gene into cultivars also see Fig. 1). Retarded activity of the CPD photolyase by way of engineering may ameliorate UV sensitivity, in Norin 1 compared with that in Sasanishiki was dem- thus leading to substantial increases in the productivity onstrated using crudely extracted enzymes from cultivars of economically important crops. The biochemical char- (Hidema et al., 2000; Teranishi et al., 2004). The results acterization of rice photolyases should therefore be help- of the present study indicate that the Norin 1 enzyme can ful for the development of improved rice cultivars. complement phr-defective E. coli and bind UV-damaged DNA in vivo in the dark (Fig. 3), bind CPD in vitro (Fig. This work was supported by Grants-in-aid for Scientific 5), and bind FAD (Fig. 7) as efficiently as the Sasanishiki Research from the Ministry of Education, Culture, Sports, Sci- enzyme. The absence of any difference between the ence and Technology, Japan (13876073 to K. Y., 14704060 to J. H. and 12480154 and 15201010 to T. K.). Sasanishiki and Norin 1 enzymes in this study is likely because of the usage of E. coli cells. As mentioned above, the Arabidopsis class II photolyase prepared from E. coli REFERENCES contains FAD and has photoreactivating ability, but a Ahmad, M., Jarillo, J. A., Klimczak, L. J., Landry, L. G., Peng, second chromophore was not detectable (Kleiner et al., T., Last, R. 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