Mechanistic Insights Into Dimerization Of Pif1 Helicase

Helicases are ubiquitous in eukaryotes,prokaryotes,and viruses.They play critical roles in every pathway of nucleic acid metabolism,and is involved in mediating a variety of cellular stress responses,including apoptosis,aging,and autophagy,thereby helping cells deal with replication errors and endogenous or exogenously triggered DNA damage.In addition,deficiency in the helicase-dependent mechanisms will cause a series of problems such as the aging of organismal levels and tissue,and neurological system defects.As an ATP-dependent SF1 B helicase,Pif1 was discovered for the first time due to its important role in maintaining the stability of mitochondrial DNA in yeast.Nowadays Pif1 has been found in all the eukaryotes and some bacteria.There have been many reports about that Pif1 helicase being involved in various important physiological activities,including the maturation of Okazaki fragments,the replication of ribosomal and mitochondrial DNA,and the regulation of telomeres.In addition,highly stable G-quadruplex(G4)structure formed in the guanine-rich chromosomal region is a key factor in replication hindrance and DNA break damage.Studies have shown that Pif1 bind tightly to the G4 structure.Moreover,comparing with other helicases,Pif1 exhibits higher specificity and unwinding activity for the G4 structure.Therefore,Pif1 play critical roles in cells due to its specificity for G4.Several studies have shown the DNA induced dimerization of Pif1 helicase,however,lack of Pif1 dimeric structure makes it difficult for us to clarify the regulation mechanism of dimerization on Pif1 helicase activity.Therefore,prokaryotic Themus oshimai Pif1(ToPif1)from thermophilic bacterium was selected for structural and biochemical study.Moreover,the structural mechanism of ToPif1 dimerization at the atomic level was revealed by single crystal diffraction and small-angle scattering techniques.Besides,the aggregation states of ToPif1 were characterized by the size exclusion chromatography and dynamic laser-based scattering technology,and then the stopped-flow assay and smFRET based on the energy transfer of resonance fluorescence were used to systematically interpret the dynamic mechanism of Pif1 dimerization.The main results are as follows:1.We have obtained the crystal structures of ToPif1 in apo state,complexed with the partial duplex,and the dimeric structures of ToPif1 compounded with ssDNA for the first time.Thus,we have derived the structural information of ToPif1 in different states.2.Accordingly,in terms of overall structure and individual domains,ToPif1 monomer is highly similar to the structures of other Pif1 helicase including Ba Pif1(PDB code: 5FHG),Bs Pif1(PDB code: 5FTD)and human h Pif1(PDB code: 6HPU).In the ToPif1-apo monomeric structure,domain 2B is stabilized in a closed conformation by interacting with both domains2 A and 1A,occluding the ssDNA binding site.Furthermore,ToPif1 helicase is inactivated due to the closed conformation of domain 2B by intramolecular crosslinking.3.Partial duplex DNA binding causes the domain 2B of ToPif1 to switch from closed to open conformation.Unfortunately,electron densities corresponding to the ds DNA are not strong enough for determining their structures,suggesting that these structures are statically or dynamically disordered.Therefore,the small-angle scattering data supplements the ds DNA structure.Besides,fluorescently labeled ToPif1 protein is used to further reveal the potential ds DNA unwinding mechanism of ToPif1 at the single-molecule level.Unlike other SF1 A helicases that unwinding in an open or closed conformation,the ToPif1 helicase mechanism is correlated with repetitive movement of domain 2B between opening and closing states.4.We have observed dimeric structures of ToPif1 with ssDNA.According to the comparative analysis of all the crystal structures,we find that the ssDNA binding allows both ToPif1 molecules to be maintained and locked in a more open conformation for these dimeric structures.Therefore,we speculate that the negative regulation mechanism of dimerization on ToPif1 helicase activities.Finally,we prove that ToPif1 dimerization induced by ssDNA binding in solution state result in an auto-inhibitory effect on its helicase activity.In conclusion,our study reveals the unwinding mechanism of ToPif1 and negative regulation mechanism for ToPif1 dimerization in terms of structure and helicase activities.According to structures of ToPif1 and activities of mutants,conformational changes of the 2B domain are important for Pif1 helicase.The structural mechanism proposed in this paper can be used as a new direction for studying how dimerization affects/regulates DNA replication,recombination and repair,and provides a structural basis and new enlightenment for the potential physiological significance of dimer regulation.Therefore,our study help people to study the function of Pif1 helicase in more depth and is helpful for further studies about SF1 B helicase function.Finally,this work may shed new enlightenment on the structural basis for dimerization and regulation mechanism in different helicases.