Biochemical Properties And Active Sites Of Thermophilic Pif1 Helicase

The Pif1 protein family is a G4-specific helicase in superfamily I,which is widely found in viruses,prokaryotes and eukaryotes.Pif1 helicases play multiple functions during cellular metabolism,including mitochondrial DNA maintenance,negative regulation of telomerase,involvement in Okazaki fragment processing,and synergy with the polymerase Polδ to stimulate repair-related DNA synthesis.In addition,Pif1 can suppress genomic instability due to G4 motif through efficient G4 unwinding activity,preventing cells from phenomena such as replication fork damage,aberrant epigenetic silencing,and total chromosomal rearrangement.So far,it has found that Pif1 has diverse ds DNA unwinding mechanisms in different species,for example,Pif1 in Saccharomyces cerevisiae unwinds ds DNA in dimers,while ToPif1 in thermophilic bacteria exerts ds DNA unwinding in monomeric form.However,the current research on the unwinding mechanism of G4 DNA structure by Pif1 mainly focuses on Sc Pif1,and the understanding of the G4 unwinding mechanism of other species Pif1 is lacking.In addition,considering that the thermostability of helicase affects its interaction with G4 under physiological conditions at higher temperatures in vivo,the thermophilic bacteria Sulfurihydrogenibium sp.YO3AOP1 Pif1(SusPif1)and Themus oshimai Pif1(ToPif1)were selected as the research object.First,the SusPif1 protein was expressed by prokaryotic system,and purified by Ni-NTA column and Hi Trap SP HP column to obtain the target protein with high purity.Then the binding activity and unwinding activity of SusPif1 helicase to ds DNA and G4 substrates was detected by fluorescence anisotropy assay and fluorescence resonance energy transfer coupled with stopped-flow FRET technology(stopped-flow FRET),and the thermophilic properties of SusPif1 and ToPif1 proteins were characterized by circular dichroism and stopped-flow FRET techniques.Then,in order to study the interaction between thermophilic bacteria SusPif1 with G4 at the structural level,the composite crystals of SusPif1 with different types of G4 DNA were initially screened.But unfortunately,the high-resolution SusPif1-G4 composite crystal structure was not obtained.Therefore,we choose ToPif1 whose crystal structure has been resolved as the material to study the interaction between thermophilus Pif1 with G4.The structure of ToPif1-G4 complex was simulated by molecular dynamics,and the key amino acid sites affecting the binding and unwinding of ToPif1 to G4 with different configurations were analyzed by using point mutation,combined with ATP hydrolysis test,fluorescence anisotropy test and stopped-flow FRET technology.The following conclusions were reached:(1)The binding assay showed that SusPif1 helicase prefers to bind ds DNA and G4 substrates with longer tails;through unwinding experiments,it was found that tails with a length of ≥18nt and G4 structures can effectively promote the unwinding of downstream ds DNA by SusPif1.(2)Both the structures of SusPif1 and ToPif1 have strong heat resistance,and at 50°C high temperature,SusPif1 will lose its unwinding activity,while ToPif1 still retains a certain unwinding activity,indicating that ToPif1 has the unique thermophilic properties.(3)Eight potential sites of interaction with G4 on ToPif1 were determined by molecular simulation,the corresponding single-point mutant protein was tested by ATP hydrolysis,and the single-point mutation of the eight sites did not destroy the basic activity of ToPif1.(4)When ToPif1 helicase with thermophilic properties binds G4 DNA with different configurations,the amino acids that play a key role are different,and the R355 position is crucial for the binding of parallel and antiparallel configuration G4.Importantly;the key active sites in ToPif1 involved in unwinding parallel and antiparallel G4 DNA are R135 and R355.In conclusion,this thesis focuses on the thermophilic bacteria SusPif1 and ToPif1 helicases,elucidated the substrate binding and unwinding properties of SusPif1 in vitro,and characterized the thermophilic properties of SusPif1 and ToPif1 proteins.The key amino acid positions of ToPif1 involved in binding and unwinding of G4 DNA were clarified,which provided a reference for in-depth understanding of the mechanism of G4 recognition and unwinding of Pif1 in thermophilus.