Structural Basis For DNA Unwinding At Forked DsDNA By Two Coordinating Pif1 Helicases

Pifl family is a group of DNA helicase which has been identified in all eukaryotes,some prokaryotes and viruses.It can unwind DNA in the direction of 5'-3',belonging to superfamily 1(SF1B)helicase.Pifl uses the energy produced by hydrolyzing ATP to unwind dsDNA or disrupt stable nuclear protein complexes to maintain the genomic stability of the nucleus and mitochondrial,including Okazaki fragment processing,telomere homeostasis,and unwinding G-quadruplex structure.Pifl plays a number of roles in maintaining genomic stability and preferentially unwinds forked dsDNA,but the molecular mechanism by which Pifl unwinds forked dsDNA remains unclear.In this study,we solved the crystal structure of Bacteroides sp Pifl(BaPifl)in complex with a symmetrical dual forked dsDNA.We found that two coordinating BaPifl molecules bind to the single strand DNA of each forked dsDNA,and interact with the 5' arm and 3'ss/dsDNA junction regions,respectively.Each of the two BaPifl molecules has helicase activity,and the interaction between them may regulate their helicase activities.The structure reveals several important features that distinguish BaPifl from any other known SF1 and SF2 helicases.First,the BaPifl molecule bound to 5' arm breaks the first base-pair,which initiates the unwinding of the forked dsDNA by BaPifl,while the BaPifl molecule bound to 35 arm plays an accessory role in the unwinding process by stabilizing the first broken base-pair and engaging the second base-pair in a pre-breaking state Second,the BaPifl molecule bound to the 3' arm prevents the annealing of the displaced strand and the tracking strand.Subsequently,two BaPifl molecules contact with each other through electrostatic interactions,which may allow the two BaPifl molecules to regulate each other's activity.Furher supports for this notion come from kinetics and single-molecule fluorescence resonance energy transfer(sm-FRET)analysis.In summary,our crystal structure combined with kinetics and sm-FRET data advance our understanding on the molecular mechanism governing forked dsDNA unwinding by BaPifl.The results presented in this thesis not only rationalize the previous biochemical results,but also provide a clue for further elucidating the mechanism by which Pifl helicase unwinds RNA/DNA hybrids and G4 DNA.