New Insights Into Strand Displacement And Unwinding Behavior Of Klenow Fragment Revealed By SmFRET Technique

This study was carried out to elucidate the mechanism of strand displacement and unwinding behavior of Klenow fragment(exo-)in GC-rich DNA sequences through singlemolecule FRET.GC-richness of a DNA possesses the redundancy of the Guanine(G)and Cytosine(C)bases in its both strands.We find that this property has influence on the strand displacement phenomenon for Klenow fragment.We observe numerous backward steps after the incorporation of dNTP in presence of KF(exo-).We also find that less backward steps occur in the strand that consists of low GC-rich sequences or AT-rich sequences.Thus,we suggest,this phenomenon represents the occurrence of backward steps more in GC-rich DNA than in AT-rich DNA.Using different concentrations of dNTP gives a new insight into this process.Increasing the dNTP concentration leads to the occurrence of less backward steps.This may represent the concentration dependence of the strand displacement,which is previously unobserved.We also monitor the unwinding step size by Klenow fragment and find that,at the presence of dNTP,it can unwind with a step size of 1 to 2 base pairs.But the probability of the unwinding ability of 2 base pair step size increases with the elevation of dNTP concentration.These two results reveal not only the importance of the GC-richness but also the influence of dNTP concentrations in the strand displacement of KF.