The DNA Photolyase Family And Their Repair Mechanism Under Ultraviolet Radiation In Antarctica Algae Chlamydomonas Sp.ICE-L

CPD and 6-4PPs are UV-induced DNA lesions.DNA Photolyases,a class of flavoproteins that utilizes energy of light,are capable of repairing DNA damage.DNA photolyase recognizes and binds to DNA lesions: CPDs or 6-4PPs and photolyase usually classified as CPD photolyase or(6-4)photolyase.The Antarctic microalga,C.sp.ICE-L,can thrive in extreme environments of the Antarctic.DNA photolyases are essential for the Antarctic microalga survival under intense UV radiation in Antarctica.This paper is a preliminary research on the photolyases family of C.sp.ICE-L.A CPD photolyase have been iditified from C.sp.ICE-L before.From the transcriptome of C.sp.ICE-L,a(6-4)photolyase and other photolyases were found.Molecular biology was carried out and activity assay of(6-4)photolyase was performed.What's more,biplasmid co-expression system of CPD photolyase and(6-4)photolyase of C.sp.ICE-L was constructed for the first time.Objective:In this study,the genes of photolyases family from C.sp.ICE-L were cloned;the expression system was constructed and recombinant protein was expressed and purified;in vivo and in vitro assays were performed;the key amino acid of 6-4CiPhr was mutated;interaction effect of CPD photolyase or(6-4)photolyase was studied.Methods:1.The CPD formation under UV was measured by Enzyme Linked Immunosorbent Assay.2.RACE-PCR and PCR was performed to obtain cDNA of photolyases from C.sp.ICE-L.3.The transcriptional levels of photolyases were studied by quantitative real-time PCR.4.For recombinant protein expression and purified,the expression system was constructed.The biomass of recombinant cell and the concentration of recombinant protein of were optimized.5.In vitro assays were performed by UV absorption spectroscopy.In vivo assays were performed through repair-deficient E.coli strain.The photolyase activity was calculated by survival rate under UV.6.Site-directed mutation was performed in 6-4 photolyase.E193(Glu)was changed to Asp,designated E193D;W295(Trp)was changed to Cys,designated W295C;H367(His)was changed to Gln,designated H367 Q.The mutated recombinant proteins were expressed and purified.The activity assay of mutated(6-4)photolyase was performed.7.To study interaction effect of CPD photolyase and(6-4)photolyase,two-plasmid co-expression system was constructed.The expression vector of CPD photolyase and(6-4)photolyase took different antibiotic screening markers to screen the two-plasmid co-expression cell.Results:1.The C.sp.ICE-L could adapt UV-B radiation.However,compared to the control,the growth rate was inhibited by UV-B.In C.sp.ICE-L,CPD was accumulated under UV-B.The result showed UV-B induced DNA lesions in C.sp.ICE-L.2.In C.sp.ICE-L,4 putative photolyase sequences were cloned,designated 6-4CiPhr,CiPhr1,CiPhr2,CiPhr3.Bioinformatics analysis was performed.3.The transcriptional levels of 6-4CiPhr were induced by UV-B and blue light.The expression of CiPhr2 as well as CiPhr3 was up-regulated under blue light.It seems that these three proteins were blue light recepetor.Under red light,CiPhr1 underwent an active stage,indicating a red light recepetor in C.sp.ICE-L.4.The expression system of 4 putative photolyases was constructed.The recombinant protein 6-4CiPhr and CiPhr1 was purified successfully.The dry biomass of recombinant cell and the concentration of recombinant protein of were optimized.5.In vitro and in vivo assays showed the repair activity of 6-4CiPhr and CiPhr1.The 6-4CiPhr was the first(6-4)photolyase found in C.sp.ICE-L.And CiPhr1 was the second CPD photolyase found in C.sp.ICE-L.6.Site-directed mutation was performed.The repair activity of TB-E193 D decreased,while that of TB-W295 C changed little.However,TB-H367 Q lost the repair activity.7.Two-plasmid co-expression of CPD photolyase and(6-4)photolyase survived the repair-deficient E.coli from UV.Conclusions:UV-B induced DNA lesions in C.sp.ICE-L.CPD was formed and accumulated.4 putative photolyase sequences were cloned,designated 6-4CiPhr,CiPhr1,CiPhr2,CiPhr3.What's more,the expression system of 4 putative photolyases was constructed.The 6-4CiPhr and CiPhr1 were purified successfully.The result indicated 6-4CiPhr was blue light recepetor with activity of repairing 6-4PPs.CiPhr2 and CiPhr3 were putative blue light recepetor.CiPhr1 was CPD photolyase with activity of repairing CPD.Site-directed mutation of 6-4CiPhr discovered that H367 was a key amino acid.When it was changed to Gln,6-4CiPhr lost the repair activity.Two-plasmid co-expression of CPD photolyase and(6-4)photolyase was more conducive to the repairing DNA leisions and surviving the repair-deficient E.coli from UV.