Studies On The Structural Basis Of Pif1 Family's Unwinding Mechanism

Pifl is a member of superfamily 1B(SF1B)DNA helicases,playing a very important role in regulating many biological processes in cells through unwinding double stranded DNA(dsDNA),DNA/RNA hybrid and G4 DNA by using the energy of ATP hydrolysis.By unwinding the template RNA of telomerase from the telomere,Pifl displaces telomerase to control the length of the telomere.In the process of replication and translation,Pifl resolves the G4 structure rapidly to maintain the stability of genome.Pifl also participates in okazaki fragment maturation and promotes break-induced replication(BIR).Moreover,Pifl is very important in the replication process of ribosomal DNA(rDNA)and the maintenance of mitochondrial genome stability.However,it is unclear how the Pifl protein utilizes the energy of ATP hydrolysis to exert it's helicase activity.In this study,full length Saccharomyces cerevisiae Pif1(ScPif1),human Pif1(hPif1),Bacteroides sp Pifl(BaPif1)and the helicase domain(HD)of ScPif1 and hPifl were expressed and purified mainly using the E.coli expression system.The crystal structures of BaPifl,hPifl-HD,BaPifl/dT10 complex and BaPifl/dH complex were solved.By comparing the native structure with the complex structures,we found that BaPifl binds to DNA and undergoes large conformational changes with the 2A and 2B domain rotating 20° and 50° respectively.This conformational change is essential for Pifl to perform its helicase function.The cocrystallization of BaPifl with a tailed dsDNA dH resulted in a bound ssDNA bent nearly 90° at the ssDNA/dsDNA junction which would facilitate the unwinding of dsDNA.Further analysis by the combination of biochemistry,biophysics,molecular and cell biology methods,reveals the role of signature motif which is unique to the Pifl family playing in the process of unwinding.Based on these results,we proposed a possible unwinding model of BaPif1.This study not only offers an important evidence for Pifl's function in the maintenance of genomic stability,but also provides new targets and ideas for designing anti-tumor drugs.