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Biochemical and Biological Properties of DNA Photolyases Derived from Utraviolet-Sensitive Rice Cultivars

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Biochemical and Biological Properties of DNA Photolyases Derived from Utraviolet-Sensitive Rice Cultivars Genes Genet. Syst. (2007) 82, p. 311–319 Biochemical and biological properties of DNA photolyases 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 enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We 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 pyrimidine dimer (CPD), DNA photolyase, Oryza sativa, ultraviolet class II photolyases are found in some bacteria, archea, INTRODUCTION plants, 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 cofactor 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, Plant 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-transferase 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.
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