The Stepping Kinetics Of Helicases Studied With High Resolution Single Molecule Fluorescence Resonance Energy Transfer

Compared with the ensemble methods,single molecular techniques can detect the mechanism or dynamic at single molecule level.Single-molecule FRET is one of the single molecular methods,and has been widely used to study helicases.However,the necessary 1 base pairs(1 bp)resolution for the study of the helicase stepping kinetics was unrealized.To solve the problem,we designed a nanotensioner,by making the DNA longer and more stable,we increased the resolution of single molecule FRET to 0.5 bp and elevated the study of helicase to a new level.Furthermore,this technique could be useful on studying other motor proteins,such as polymerases,which unwind double-stranded DNA into single-stranded DNA with higher precision.Based on this high resolution sm FRET,I proceed on several helicase stepping studies as below.Helicases hydrolyze NTP to uncoil double stranded DNA step by step.Although there have been a lot of research about that,one still lack the understanding of the stepping process.Using single-molecule FRET,we studied two kinds of non-cyclic helicases,Pif1 and RecQ.The data reveals that the steps of the both helicases are ununiformed,and RecQ is different with Pif1 about force responding.The phenomena that Pif1 stepping is force sensitive is maybe because of its special function in cells.Furthermore,the ununiformed helicase stepping requires some kind of structural basis.Unfortunately,there was no detailed discussion on the corresponding structural basis of this stepping.In order to study the structure foundation,we used BLM helicase and proceeded some high precision single molecule FRET experiments.We found that the BLM-DNA complex exists several stable complex structures,which serve as the structural basis for the ununiformed stepping.When we repeated the experiments with RecQ,we found similar results as BLM.These results indicate these structural characteristics may be kind of universal.Lastly,based on our high-precision stepping data for several helicases,by introducing two dynamic parameters to characterize the random release processes quantitatively,we built a unified quantitative unwinding model.This model can quantitatively explain the unwinding pattern we observed and provide a unified model for the helicase stepping process.