From Thermal Instability To Light Instability:The Properties Of Photolyase And Its Relationship To The Molecular Evolution Of Cryptochrome

Photolyases are a class of enzymes that directly repair the UV-induced lesions in DNA.They can be divided into two types: CPD photolyases and 6-4 photolyases,which repair the two major UV-induced lesions,cyclobutane pyrimidine dimers(CPDs)and 6-4 photoproducts in DNA,respectively.Photolyases can catalyze the repair reaction to restore the normal structure of DNA using 315-500 nm light as an energy source.Cryptochromes are homologs of photolyases,but they have no DNA repair activity.Cryptochromes interact with their partners to regulate expression of target genes.Plant cryptochromes play major roles in plant development and photomorphogenesis,and possibly act in the entrainment of the plant circadian rhythm.The main function of animal cryptochromes is to maintain the animal circadian rhythm(play role in circadian clock).Although the photolyases and cryptochromes have diverse functions,their sequences and structures share a high similarity.All of them can bind the cofactor FAD,and play functions through the common mechanisms.Thus,all of these proteins are termed as cryptochrome/photolyase family(CPF).The properties of Escherichia coli photolyase were studied in vivo and in vitro.As the growth temperatures ? 37°C,the activity of E.coli photolyase in vivo was decreased dramatically,and the protein level of photolyase was also decreased.However,the levels of phr transcripts(encoding photolyase)were almost unchanged.A lacZ-reporter under the transcriptional control of phr promoter showed no temperature-dependent expression.However,a translational reporter of the photolyase-LacZ fusion protein exhibited lower ?-galactosidase activity at high growth temperatures.These results indicated that the decrease of photolyase activity at different growth temperatures is post-transcriptional in nature.The possible cleavage sites in E.coli photolyase by proteolysis were determined,and the results indicated that the apo-form photolyase seems to have more sensitive sites for cleavage by proteases.In vitro,assays revealed that the denaturation of active photolyase occurs under temperatures ?37°C,while apo-photolyase unfolds under temperatures ?25°C.The temperature-shift experiments showed that the active photolyase is relatively stable in vivo,but newly synthesized apo-photolyase is more labile to thermal unfolding and proteolysis.These results suggest that thermal unfolding and proteolysis of newly synthesized apo-photolyase are responsible for the impaired photolyase activity in vivo at high growth temperatures.The finding has some practical implications for UV disinfection.The proper doses of UV may be strategically applied to prevent photoreactivation at different ambient temperature after UV irradiation.In this study,we found that E.coli photolyase is a thermal unstable protein.The thermal instability of E.coli photolyase may play roles in environmental adaptations and evolution of E.coli.The thermal instability of photolyases may also provide a prototype for the evolution of light labile cryptochromes.The phylogenetic analysis of the possible CPF protein sequences derived from the whole-genome and the transcriptome sequences in NCBI database was conducted.According to the phylogenetic tree,the CPF proteins can be divided into three main groups: CPD class I photolyase related proteins,CPD class II photolyases and FeS-BCPs(Fe-S bacterial cryptochromes and photolyases).CPD class II photolyases are mostly found in archaebacteria and eukaryotic organisms.FeS-BCPs are a new CPF group,most of which containing iron-sulfur clusters.FeS-BCPs are possible prokaryotic 6-4 photolyases,and may be the common ancestors of CPF proteins.CPD class I photolyase related proteins can be further divided into CPD I/CPD III/plant CRY,DASH/6-4 photolyase/animal CRY/GIHY,and SPL/MPL proteins.The branch of CPD I/CPD III/plant CRY contains CPD class I photolyases and CPD class III photolyases,and plant CRYs are clustered into the sub-branch of CPD class III photolyases.The branch of DASH/6-4 photolyase/animal CRY/GIHY is consisted of DASH proteins,6-4 photolyases and GIHY proteins.DASH proteins are probably single-stranded CPD photolyases.6-4 photolyases in this branch are mostly found in eukaryotic organisms.Animal CRYs are clustered into the sub-branch of 6-4 photolyases.GIHY proteins are a newly named sub-branch in this study,which may be prokaryotic cryptochromes.SPL/MPL proteins discovered in this study make a new branch of CPD class I photolyase related proteins.They are probably CPD photolyases,but shorter in length(200-400 aa),which lack the N-terminal homological sequences of normal photolyases and cryptochromes.Some of SPL/MPL proteins also contain iron-sulfur clusters.They may be the alternative candidates of the common ancestors of CPF family.Based on the phylogenetic analysis,we found that the residues at the site near the pyrimidine ring of isoalloxazine moiety of FAD(A377 in E.coli photolyase)are significantly different in photolyases and cryptochromes.In CPD class I,class II and class III photolyases,DASH and SPL/MPL proteins(which may use CPD as substrate),the residues A,S and N frequently occur in this site.In 6-4 photolyases and FeS-BCPs(which may use 6-4 photoproduct as substrate),and animal CRYs and function-unknown GIHY proteins,the residues I,L and V frequently occur in this site.In plant CRYs,most of them has S in this site and the rest of them has C in this site.To elucidate the possible functions of this site,several mutants with the replacement of A377 in E.coli photolyase by the residues S,N,I,L,V and C were constructed.The mutant proteins were expressed and purified.The catalytic activity,spectroscopic properties,the light stability and the stability toward oxidation were analyzed.The mutant proteins had normal catalytic activity as compared with the wild type photolyase,indicating that this site has little effect on catalysis.A377 S and A377 N mutants had a small blue shift in the absorption spectra and were more stable under light illumination when compared to the wild type.The oxidation rates of A377 S and A377 N mutants were significantly retarded.However,A377 I,A377V and A377 C mutants had a large red shift in the absorption spectra and were unstable under light with the release of FAD cofactor during illumination.These three mutant proteins were oxidized much faster in aerobic conditions.The results indicated that the replacement of A377 with I,V,C residues caused E.coli photolyase more unstable under light,which are similar to some cryptochromes.Therefore,the change of residues at A377 site may be an important intermediate process of evolution from photolyases to cryptochromes.