Probing Helicase Dynamics On Nucleic Acids Through Fluorescence-Force Measurements

Helicases are crucial enzymes which regulate metabolism of nucleic acids and have been studied extensively at biochemical, structural and genetic levels. Yet limitations of experimental approaches restrained full characterization of helicase activities. Here, we have applied hybrid platforms of single-molecule fluorescence detection combined with optical tweezers under TIRF or confocal configurations to reveal new properties of nucleic acids translocation and unwinding activities of helicases.With aid of mechanical manipulation, we probed the translocase activity of hepatitis C virus NS3 helicase on laterally stretched, kilobase-size single-stranded DNA and RNA. The results demonstrate that NS3h monomer is a highly processive ssDNA and ssRNA translocase with force-independent translocation rates. Moreover, NS3h translocation rate is three times faster on ssRNA compared to ssDNA, which can be explained by differences in X-ray crystal structures of NS3 in complex with DNA and RNA. We observed a new translocation mode of NS3h on ssDNA, and together with smFRET experiments, we could assigned the mode to “NS3h-mediative repetitive looping”. We have showed that the looping rate is force-dependent.We are also examining the Pif1 helicase and its unwinding activities of G-loop, a three stranded structure composed by G-quadruplex and RNA-DNA hybrid structures. Recent studies suggested that G-loop of hexanucleotide expansion repeats at C9orf72 gene is highly associated with neurodegenerative diseases. Our results show that Pif1 monomer can unwind HRE-G4, which has been proposed as a four-layer G-tetrads, in a discrete, stepwise manner. Other data indicate that self-hairpin structure is predominant at low ionic strength, and HRE-G4 structure is predominant when ionic strength is higher. With the characterization of structural features of HRE-G4 and unwinding mechanism by Pif1, we can begin to address the question of how the folding of HRE-G4 affects the unwinding of RNA-DNA hybrid on HRE G-loop.