T7 helicase unwinding and stand switching investigated via single-molecular technology

Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers are widely used to study the molecular motors because of their high resolution and real-time observation. In this work, we choose these two techniques as the research means. The bacteriophage T7 helicase, as the research object, serves as a model protein for ring-shaped hexameric helicase that couples deoxythymidine triphosphate (dTTP) hydrolysis to unidirectional translocation. The DNA strand separation is 5'-3'-along one strand of double-stranded DNA. Using smFRET and magnetic tweezers to study the unwinding process of T7 helicase, we can have more in depth understanding of the unwinding and strand switching mechanisms of the ring-shaped hexameric helicases. First, by designing DNA substrates with different 3'-tail structures, we find that the 3'-tail is required for T7 helicase unwinding process, no matter whether it is single-stranded or double-stranded. These results confirm an interaction between T7 helicase and 3'-tail. Second, examining the dependence of unwinding process on GC content in DNA sequence, we find that as GC content increases, T7 helicase has higher chances to stop and slips back to the initial position by annealing stress or dissociating from DNA substrate. As the GC content increases to 100%, 79% helicases could not finish the unwinding process. Third, by further analysing the experimental data, two different slipping-back phenomena of T7 helicase are observed. One is instantaneous and the other is slow. The results from the experiment on magnetic tweezers also confirm this slow slipping-back phenomenon. This instantaneous slipping-back results from the rewinding process of unwound single-stranded DNA as studied previously. When T7 helicase cannot continue unwinding because of the high GC content in DNA sequence, it dissociates from the single-stranded DNA or slips back to the initial position very quickly because of the annealing stress. However, this slow slipping-back phenomenon cannot be explained by this reason. According to previous researches, T7 helicase can only be translocated or unwound from 5' to 3' along one strand of double-stranded DNA because of the polarity principle. We suggest that this slow slipping-back is induced by the strand switching process of T7 helicase. Through this strand switching process, T7 helicase binds to the 3'-strand and are translocated along it from 5' to 3' to the initial position, results in this slow slipping-back phenomenon. This is the first time that the slow slipping-back phenomenon has been observed, which strongly suggests the strand switching process of T7 helicase. Based on our results and previous researches, we propose the model of this strand switching process and this model may be extended to all ring-shaped hexameric helicases.