A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification

Abstract

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

“…Bacterial strains, plasmids and media The E. coli strains NKJ3002 (phr -uvrA -recA -) (Nakajima et al, 1998), KY20 (phr -) (Hirouchi et al, 2003) and DH5α (phr + recA -) (Grant et al, 1990) were used as hosts for the cloning of rice photolyase genes, overexpression and purification of GST-photolyase fusion proteins, and complementation of the rice photolyase gene in vivo. The plasmid pGEX-4T used for the glutathione-S-transferase (GST)-fused constructs was purchased from Amersham Biosciences.…”

“…We have recently characterized a class II CPD photolyase from Oryza sativa (Hidema et al ,2000;Hirouchi et al, 2003;Teranishi et al, 2004). It has been indicated that Asian rice cultivars differ in their response to UV radiation in terms of growth and physiological processes (Teramura et al, 1991).…”

Ultraviolet-Defense Mechanisms in Higher Plants

“…these two classes of cPD photolyases share only 10% to 15% sequence similarity (75). homologs of cPD photolyases and/or 6-4 photolyases have been cloned from various plant species (1,23,54,58,65,76). characterization of laboratoryinduced UV-sensitive mutants has been a powerful tool for studying photorepair in plants.…”

“…it was demonstrated that the photolyase-CPD complex in "Norin 1" was highly thermolabile compared with the photolyase in "Sasanishiki" (20). A cDNA clone of CPD photolyase in "Sasanishiki" was isolated (23). As shown in Fig.…”

UV Damaged DNA Repair & Tolerance in Plants

“…Genes encoding CPD photolyases and 6-4 PP photolyases have been identified and characterized in a range of prokaryotic and eukaryotic systems (Sancar, 2003). In plants genes encoding CPD photolyases have been identified in Arabidopsis thaliana (Ahmed et al, 1997), cucumber (Takahashi et al, 2002), rice (Hirouchi et al, 2003), spinach (Yoshihara et al, 2005), and soybean (Yamamoto et al, 2008). Genes encoding 6-4 PP photolyases have been identified in Arabidopsis and rice (Chen et al, 1994;Singh et al, 2010).…”