Investigate Pif1 Helicase And Shelterin Against RPA By SmFRET

Single-molecule Fluorescence Resonance Energy Transfer(smFRET)is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA.However,due to the thermal fluctuation and random disturbances,smFRET has been lacking single-base pair(1-bp)resolution required for revealing stepping kinetics of helicases.The resolution is limited to 2-3 bp.We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs to achieve one-nucleotide resolution fluorescence resonance energy transfer and observe Pifl helicase one base pair stepping.We speculate that the nanotensioner technique will find wide applications in smFRET studies of many other motors that convert dsDNA to ssDNA and vice versa such as helicases and polymerases.Pifl is a multi-functional helicase,which plays important roles in DNA replication processes such as Okazaki fragment maturation(OFM),break-induced replication(BIR)and the maintenance of telomeric DNA and mitochondrial genome stability.Therefore,to research Pifl unwinding mechanism is of vital importance.We found that Pifl was actually a monomeric helicase.A Pifl monomer may unwind a forked DNA repetitively for many unwinding-rezipping cycles.However,it has a very limited processivity(?10bp)in each cycle because it releases the tracking strand readily.With increasing force,the unwinding rate and processivity of Pifl increase significantly.We think that this special character is relevant to its cellular functions.On the other hand,the high resolution Pifl stepping data reveal that Pifl exhibits 1-bp-stepping kinetics but sequesters the nascent nucleotides and releases them randomly.The unprecedented resolution provided by presented nanotensioner approach enabled us to reveal the details of Pifl helicase-catalyzed DNA unwinding that are not easy to study with conventional smFRET method.Telomeric DNA need to be protected against DNA damage repair(DDR)signals to endure genome integrity.Telomeres form a protective cap structure at chromosome ends by recruiting the shelterin complex,including TRF1,TRF2,RAP1,TESN2,TPP1 and POT1 six subunits.The protection of telomere 1(POT1)subunit protects telomere by specifically binding to the single stranded telomeric DNA.Assembly of the shelterin complex onto telomeric termini may cross-bridge the single-stranded and double-stranded telomeric DNA tracts;and provide better protection of telomeric terminus against DNA Damage Repair signals.We have established a single-molecule imaging platform to study the dynamic interactions between shelterin and replication protein A(RPA),a DDR initiation protein that binds nonspecifically to ssDNA,at telomeric terminus.Our smFRET studies show that RPA binds GQ steadily and can not be removed by excess POT1 in 150mM Na+ buffer.And POT1 alone is not sufficient to protect telomeres against excess RPA.Shleterin provides better protection than POT1 alone against RPA binding.To unfold GQ under shelterin protection requires 3-5 folds RPA than under POT1 protection.