Cloning, Functional Characterization, And Expression Of The Gene Encoding A CPD Photolyase From Dunaliella Salina

Solar UV radiation in the UV-B (315-280 nm) and UV-C (280-100 nm)spectral regions has mutagenic, carcinogenic, and lethal effects on living organisms.The major damage induced in DNA by UV radiation is cyclobutane pyrimidinedimers (CPDs) and (6-4) photoproducts (6-4PPs). CPDs constitute the major class ofthese lesions (about 75%) and the rest mainly are (6-4) PPs. Both classes of dimermay block transcription and DNA replication if left unrepaired. Organisms havedeveloped several paths, including nucleotide excision repair (NER) andphotoreactivation, to repair DNA damage and keep the integrality of geneticinformation. Photoreactivation is a light-depend way to repair UV- induced DNAdamage under the action of photolyase, i.e., CPD photolyase and (6-4) photolyase,CPD photolyase and (6-4) photolyase specifically binds to CPDs and (6-4)PPs, andrepairs them respectively.CPD and (6-4) photolyase are both absent in placental mammals includinghuman. Many organisms posses CPD photolyase (including Escherichia coli andSaccharomyces cerevisiae), while some other organisms, including some plants,posses both types of photolyase. However, both types of photolyase have been isolated from only a few organisms, including Drosophila melanogaster andArabidopsis thaliana. Two algal CPD photolyase genes have been isolated from thespecies Chlamydomonas reinhardtii and Chlorella pyrenoidosa.The material Dunaliella salina used in the experiment is a kind of unicellulargreen alga. It is highly resistant to UV radiation and can survive in Tibet-QinghaiAltiplano where UV radiation is very strong. Since our laboratory isolated the geneof (6-4) photolyase from D. salina in 2005. We searched for the presence of anothermember of photolyase/blue-light photoreceptor. Now we have cloned andcharacterized the gene of CPD photolyase from D. salina. Then we analyzed thegene using bioinformaties software, determined its function by complementationassay and its expression pattern by real-time PCR analysis.1. Cloning of full length cDNA of D.salina CPD photolyase. We cloned the EST ofD.salina CPD photolyase using degenerate oligonucleotides as primers. Then3'RACE and 5'RACE were performed to get the 3' region and 5' region of thecDNA. Finally, a full-length cDNA of 2206 bp was cloned. We named itDsPHR2.2. Bioinformatics analysis of D.salina CPD photolyase. The cDNA of DsPHR2 andthe deduced proteins are analyzed using bioinformatics software. The cDNA ofDsPHR2 was predicted to encode a protein of 529 amino acids. The deducedamino acid sequence of DsPHR2 showed a high degree of homology to otherphotolyases from Oryza sativa, Arabidopsis thaliana, Chlamydomonasreinhardtii, and Cucumis sativus.3. Ccomplementation assay of D.salina CPD Photolyase. The cDNA fragment ofD.salina DsPHR2 encoding the photolyase was assembled in the vectorpGEX-4T-1 (yielding pGEX-DsPHR2). Then the pGEX-DsPHR2 wastransfected into Escherichia coli strain SY2 (uvrA^-, recA^-, phr^-). After irradiatingwith UV, One set sample dishes were exposed to fluorescent light from whitelamps and the control set of dishes were transferred to the dark directly. The sameexperimental conditions were used with SY2/pGEX-4T-1 without DsPHR2 as the negative control. After incubation overnight, the transformants carryingpGEX-DsPHR2 showed a significant increase in survival rate after the whitelight treatment relative to no photoreactivation and negative controlSY2/pGEX-4T-1. These results indicated that the cDNA isolated from D. salinaencodes protein with photoreaetivation activity in vivo.4. Expression patterns of DsPHR2 in D. Salina under several conditions. D. salinacells were cultured to the exponential phase. Then the cultures were transferred todark condition for 24 hours and next subjected to various treatments includingwhite light, UV, high salinity, and H₂O₂. Real-time PCR indicated that thetranscription of DsPHR2 was induced by white light, UV, high salinity, andH₂O₂.We cloned the gene encoding CPD photolyase from D. Salina and finishedbioinformatics analysis. The complementation analysis showed that the proteinencoding by DsPHR2 from D. salina has photolyase activity in vivo. Real-time PCRanalysis indicated that the mRNA synthesis of the PHR2 gene could be induced byUV-C, white light, high salinity, and H₂O₂.