Structural Studies Of Sak From Staphylococcus Aureus Phage 80? And Purification Of Photolyase Phr-related Proteins

Part I:Structural studies of Sak from Staphylococcus aureus phage 80?Sak protein is first identified and named in Lactobacillus phage ul36 and forms a Sak family with other phage-derived recombinases.ul36 Sak is the only Sak family protein that has been structurally resolved.The results of electron microscopy negative staining indicates that it shapes as an 11-mer ring structure,similar to the N-terminal domain of human Rad52 protein,and is considered to be one of the early evolutionary proteins of Rad52.According to the sequence,structure and functional similarity,Sak family is classified as Rad52-like superfamily,and the related research of Sak family proteins is helpful to reveal the mechanism of action of Rad52-like proteins.Staphylococcus aureus is a human pathogen.Recently,in the study of the S.aureus phage 80? genome replication,80? Sak protein was identified and named.According to the sequence and functional similarity,the protein was classified into the Sak family and thought as a potential homologous of the human Rad52 protein.Rad52 is one of the core proteins of double-stranded DNA breaks(DSB)repair,and participate in the homologous recombination repair of DSB in both Rad51-dependent and Rad51-independent manners.To elucidate the specific mechanism of action of Rad52,the N-terminal of human Rad52 and its crystal structure with the nucleic acid complex were resolved.The results showed that Rad52 has an 11-mer cyclic structure in solution,which is hydrophobic inside and hydrophilic outside and it contains two DNA binding sites.The single-stranded DNA of the random conformation first binds to the outer binding site,opens the ?-clip under a certain force,and slides down to the inner binding site.At this position,Rad52 interacts with the nucleic acid phosphate backbone to form a B-type structure that facilitates pairing,completing the annealing reaction.The results of cryo-electron microscopy are consistent with the 11-mer ring structure,but after the single-stranded DNA is added,Rad52 assembles to form a network of more complex rings,and its assembly mechanism has not been reported in detail.Since the human Rad52 protein has not been successfully elucidated on the assembly mechanism of the ring structure to the ring stack structure in the past 20 years,and our laboratory has long been committed to the study of Staphylococcusaureus-related proteins,we hope to give a clue on Rad52 study by investigating the mechanism of 80? Sak.In this paper,we determined that 80? Sak and human Rad52 N-terminal domain are homologous,and reconstructed the structure of 80? Sak in solution with cryo-EM.The results of remodeling indicate that 80? Sak exists as single-ring,helical-ring and double-ring conformations,and the double-ring conformation including two distinct states,namely "face to face" and "back to face".A series of biochemical experiments supported our structural visualization.Finally,we visualized the state of the 80? Sak and ssDNA complex,which revealed that double-ring structure is the basic unit for assembling stacks.In conclusion,we speculate that part of the single-ring is prone to form double-ring structures mediated by the transition state helical-ring.The two states of the double-ring could assemble to stacks even no DNA appears,and the adding of the ssDNA promotes the aggregate level of the stalks.These findings deeply proved that 80? Sak is homologous to human Rad52 N-termini in structure and shed a light to better understand the mechanism of Rad52-related proteins.Part ?:Expression and Purification of Photolyase phr-related ProteinsThe effect of ultraviolet light on biological individuals is multifaceted.Plants and autotrophs make use of it to synthesize carbohydrates and transform that into bio-energy.Humans exert sunlight to synthesize vitamin D.However,DNA is constantly attacked by ultraviolet of the solar light,which causes CPD,6-4PP and other types of DNA lesions.Photolyase system is conserved in organisms and responsible for the repair of the DNA lesions.However,in mammals like humans,there is merely conserved NER pathway for CPD and 6-4PP repair,which is not adequately efficient and leads to accumulation of the lesions and further results in dermatitis,rash,melanin and even skin cancer.Sunscreen wearing is the most common method to cope with UV damages.But there are some limitations for both organic and inorganic sunscreens,we hope to develop ways to evade sunburns and DNA damages by exploiting photolyase systems of plants or algae that showed high efficiency and accuracy for DNA repair as protective elements in sunscreens.After a series of survey and screening,we finally focused our attention on Athrospira/Spirulina platensis which has been consumed by humans for more than 300 years.Here,we expressed and purified the Ap-phr protein and its mutants from Athrospira/Spirulina platensis as well as the phr protein from And after that,we conducted the experiments on its CPD repair activity,which revealed that Ap-phr is able to repair CPD lesions both on single-strand DNA and double-strand DNA.These works provide samples to further study the mechanism of Ap-phr and lay foundations for its industrial application.