The Study On Molecule Mechanism Of Pif1 Helicase Unwinding G-quadruplexes

G-quadruplexes（G4） are four-stranded secondary structures held together by noncanonical G-G base pairs. They served a variety of cellular functions such as telomere maintenance, DNA replication initiation, and so on. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Therefore, cells have evolved highly specific helicases to resolve G4 s structures. Pif1 helicase stands out among those helicases that resolves G4 structures very effectively in vivo and in vitro. Recently, there are increasing researches to focus on the Pif1 helicases’ properties in physiological and biochemical as well as genetics fields for its excellent unwinding activity on G4 s, and these studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins（ScPif1 and hPif1）. While, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Furthermore, very little is known about the kinetics characteristics of ScPif1 catalyzed G4 s unwinding. And how this helicase resolves G4 in molecular level remains to be revealed.Accordingly, to address these three issues, we made correlated studies on the following topics. We reported the biochemical characteristics and kinetics mechanism analyses of the purified recombinant Pif1 helicase from Bacteroides spp. 3₁₂3（BsPif1）. And we used stopped-flow technology to monitor the kinetics processes of the recombinant ScPif1 protein catalyzed G4 unwinding. Besides, the single-molecule fluorescence assays were applied to the molecular mechanism of ScPif1-catalysed unfolding of G4. These studies are all aimed to clarify the kinetic characteristics and molecular mechanism of Pif1 helicase catalyzed G4 unwinding, which can provide insights for further understanding on the physiological function of the Pif1 helicase. The main results were shown as following.1, This research firstly systematically studied the biochemical characteristics and unwinding kinetics processes of the BsPif1 helicase. The results showed that: 1) BsPif1 helicase is a monomer as the unwinding active state in solution. 2) BsPif1 helicase is in accordance with some properties in all Pif1 family helicases characterized. 3) As the prototypical member of Procaryotic Pif1 helicase, BsPif1 is quite different from ScPif1 in biochemical properties and kinetic characteristics. 4) BsPif1 efficiently unfolds G-quadruplexes DNA substrates, and the kinetic parameters with its own characteristics. These results suggest BsPif1 might resolve common issues arising from during DNA transactions, and it may perform a variety of important physiological functions in vivo. Besides, these results also suggest that BsPif1 resembles more human Pif1 protein than ScPif1 with regard to substrate specificity, helicase activity and mode of action.2, During the study of Sc Pif1-mediated G4 DNA unwinding kinetics mechanism, we firstly discovered that the activation phenomenon of G4 structure on Pif1 helicase unwinding duplex DNA. The results showed that: 1) It is confirmed ScPif1 helicase can efficiently unwind G4-DNA/dsDNA and be proved energy by the ATP hydrolysis. 2) G-quadruplex DNA strongly stimulates ScPif1 helicase-mediated duplex DNA unwinding, with its own special characteristics; Pif1-mediated duplex DNA unwinding is not influenced by the presence of the G3 motif. 3) G4 s activate duplex DNA unwinding by increasing the unwinding rate, step-size and processivity.4) DNA binding properties of Pif1 under equilibrium, the binding kinetics of G4-containing partial duplex and the DLS results, all revealed that this stimulation resulted from G4-enhanced Pif1 dimerization. These results likely reflect possible regulatory role of G4 s in the rapid rescue of the stalled lagging strand synthesis.3, In this study, we used single-molecule fluorescence assays to probe molecular processes of Pif1-catalysed unfolding of G4. The results showed that: 1) Pif1 protein unfolds single G4 structures repetitively. 2)Pif1 unfolds G4 sequentially in two large steps,and the G-triplex（G3）structures play an essential intermediate in this process. 3) According to the singlemolecule FERT observation, we developed a molecule mechanism for G4 unfolding. This single-molecule mechanism model for G4 unwinding that provided not only an integrated view on how Pif1 resolves G4 structures, but also great details in spontaneous refolding and repetitive unfolding of G4. And this new-type repetitive shuttling may allow Pif1 to effectively unfold G4 and thus create more opportunities for DNA polymerase to restart stalled replication forks promptly.Taken together, by a lot of analytical methods including stopped-flow fluorescence detection, single-molecule fluorescence assay, fluorescence polarization assay and etc, we systematically investigated the kinetic characteristics and molecule mechanism of Pif1 catalyzed G-quadruplexes unwinding. This paper provides empirical guidance for the analysis of unwinding kinetic characteristics and single-molecule mechanism on other helicase with specific DNA substrates. In addition, it builds a new theoretical basis for further understanding on the relationship between structures and functions of Pif1 helicase.