New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment (exo-) of DNA polymerase I in DNA sequences rich in guanine and cytosine (GC) bases. We find that there exist in the FRET traces numerous ascending jumps, which are induced by the backsliding of Klenow fragment on DNA chains. Our measurements show that the probability of backsliding is closely related to the GC-richness and dNTP concentration: increasing the GC-richness leads to an increase in the backsliding probability, and increasing the dNTP concentration however leads to a decrease in the backsliding probability. These results provide a new insight into the mechanism of DNA polymerase I.