Study On Specific Sites Of BsPif1 Helicase Acting On G-quadruplex DNA

Helicases are molecular motors that use the chemical energy released by hydrolyzing nucleoside triphosphate?NTP?to unlock the hydrogen bond between DNA or RNA.They are involved in almost all aspects of DNA and RNA metabolism.Pif1 helicase is one member of SF1B helicases that conserved in viruses,prokaryotes and eukaryotes.In recent years,the research of Pif1 helicase have been increasing gradually,and many structures have been published,these results help us understand the working mechanism and biological function of Pif1 helicase.In 2016,Chen et al published crystal structure of BsPif1-AMPPNP,BsPif1-ADP,and BsPif1-ADP-AlF₃-ssDNA from Bacteroides spp and established the structure model of BsPif1 and dsDNA complex.However,there are still many problems to be solved.For example,as a specifically G-quadruplex?G4?DNA helicase,it is still unknown that how Pif1 helicase interacts with G4 DNA.Therefore,we hope to obtain the structure of BsPif1-G4 DNA complex,and further understand the interaction between BsPif1 and G4 DNA and the biological function of Pif1 helicase in cells.We obtained BsPif1 helicase with 95%purity through prokaryotic expression and purification,and got nucleic acid substrates with high purity and stable properties by in vitro annealing,and Mono-Q purification.Further,we screened nucleic acid substrates capable of forming a stable complex with BsPif1 by means of gel filtration chromatography and dynamic laser scattering,and then we chose two kinds of G4 DNA?7T 3G4,6T TelG4?and a dsDNA?7T11bp?with a 5'tail length of 6nt or more for complex crystallization screen.Unfortunately,after trying more than 1152 crystallization conditions,one composite crystal has not yet been obtained.Therefore,the three-dimensional structure of BsPif1-G4 DNA complex was established by molecular dynamics simulation.The point mutation experiment was used to analyze the specific site of BsPif1 acting on G4 DNA.In addition,fluorescence anisotropy,stopped-flow FRET,and single-molecule fluorescence resonance energy transfer were used to analyze the specific amino acid sites of BsPif1 acting on G4 DNA.The final conclusion is as follows:1.Fluorescence anisotropy was used to analyze the effect of BsPif1 mutations on DNA binding activity.The results showed that the five sites of K92,R120,K121,K292and K341 played a very important role in the binding of TelG4 DNA in hybrid form,while R83,K87,K92,R120,K121,K292 and K341 play a very important role in the binding of3G4 DNA in parallel form and the binding of dsDNA.2.Stopped-flow FRET was used to analyze the unwinding activity of BsPif1mutations.The results were explained by two parameters:unwinding rate and amplitude.The results indicated that R83 located in the 1B domain is a specific site for unwinding G4DNA,which has no direct effect on unwinding dsDNA;while the K341 located in the 2B domain is specific for unwinding dsDNA,which has no direct effect on unwinding G4DNA;K92,R120,K121 and K292 are important sites involved in unwinding both G4DNA and dsDNA,while R120 have a big impact on the unwinding of G4 DNA.3.smFRET was used to analyze the effect of the unwinding activity of BsPif1mutations.The results showed that R83,K92,R120,K121 and K292 are important sites for unwinding G4 DNA,which was consistent with the conclusions of the stopped-flow FRET analysis.Based on the above results,we found that R83 located in the 1B domain is a site that specifically affects the unwinding of G4 DNA,which has no direct effect on unwinding dsDNA.And the R120 site located in the 1A domain plays an important role in the unwinding of G4 DNA.At the same time,the results of point mutation experiments also prove that the BsPif1-G4 DNA complex structure model established by molecular dynamics provides an important reference value for understanding the interaction between BsPif1 and G4 DNA,which will be of great value for the subsequent study of the mechanism of BsPif1 helicase.