Structural Insight Into The Multiple Functions Of ScPif1 Helicase

Pif1 helicase,as an ATP dependent DNA helicase,is widely distributed in prokaryotes and eukaryotes.It plays an important role in the maintenance of genomic stability and regulation of nucleic acid metabolism.As a typical member of Pif1 family,Saccharomyces cerevisiae Pif1?ScPif1?helicase is one of the earliest identified and deeply studied member.In vivo,studies have shown that ScPif1 helicase is involved in the maintenance of telomere stability,maturation of Okazaki fragments,G4 DNA resolved during replication,and homologous recombination repair.In vitro,studies have shown that ScPif1 possesses both 5'-3'direction unwinding activity of double-stranded DNA,RNA/DNA hybrid and G4 DNA,the most important one of these is its efficient G4 DNA unwinding function.In vivo,G4 DNA is a major obstacle to the physiological metabolism of organisms due to its highly stability.Thus,ScPif1 plays an important role in a variety of physiological metabolic pathways through its efficient G4DNA unwinding function.At present,the research of ScPif1 was mainly focused on physiological and biochemical studies in vivo and in vitro.However,up to date,structure of ScPif1 is still unsolved due to its flexible N-and C-terminal,so structural research on ScPif1 is still limited.Considering the above deficiency,in this study we designed a truncated ScPif1protein(ScPif1^237-780)which belongs to the ScPif1 helicase core by using multiple sequence alignment and secondary structure prediction.We successfully expressed the truncated ScPif1^237-780 protein through E.coli expression system and obtained the high-purity protein which can be used in the protein crystallization assays.By using the ScPif1^237-780 protein,we got various protein-ssDNA complexes crystals and successfully resolved the structure by using X-ray anomalous scattering.This is the first 3D structure of the ScPif1 helicase core domain.Afterwards,on the basis of the crystal structure,in-depth functional mechanism study of ScPif1 helicase core protein was performed by using various experimental methods including site-directed mutagenesis,gel filtration chromatography,X-ray small angle scattering technology,fluorescence anisotropy-based DNA binding technology,Stopped-flow FRET technology and single-molecule FRET technology.The main results are described as follows:1)we firstly observed a conserved Q motif that specifically binds to ATP in the structure and confirmed that the mutation of this motif can greatly impair the ATP specific recognition function of ScPif1.2)We firstly revealed a molecular mechanism that ScPif1 can specifically binds to DNA.We demonstrated that H303 and H705 selectively recognition DNA rather than RNA by the mutation experiment.This finding structurally explains the mechanism by which ScPif1 removes telomerase in vivo.3)In this study,the structural basis of ScPif1 for guanine preference was revealed by analyzing the DNA binding sites of various G-rich DNA complex structures.Accordingly,such guanine binding preference enables ScPif1 to perform G4 DNA unwinding more effectively.4)We discovered a 2C domain with a unique folding pattern in the structure of ScPif1,which was located outside the 2B domain.Besides,it was confirmed that the domain had a regulatory effect on the unwinding and translocation activities of ScPif1.5)We successfully identified a biologically relevant dimer from the crystal structure and proposed a mechanism of G4 DNA-induced ScPif1 dimerization.6)A potential G4 DNA binding site was firstly discovered in the crystal structure,several amino acids involved in G4 DNA specific binding and unwinding activities in this site were verified by mutation experiments,which can structurally interpreted the G4 DNA specific recgonition function of ScPif1.Taken together,this paper illustrates the recognition mechanism of ScPif1 with various physiological substrates and the oligomerization state after binding to these substrates by the in-depth study of ScPif1's helicase core.It provides a theoretical basis for further understanding of the multiple functions of ScPif1 in vivo.