Structural Characteristics Of G4 DNA In Saccharomyces Cerevisiae And Its Modulation Of Pif1 Unwinding Activity

G4(G-quadruplex)DNA is a special type of DNA secondary structure whose core structure is a G-tetrad plane formed by hydrogen bonding of four guanines,the G4 structure is formed when two or more planes are stabilized by monovalent cations Na~+or K~+.The G4structure is widely distributed in the genome and plays an important role in various life activities such as DNA replication,transcription,and telomere metabolism.Therefore,proper treatment of the G4 structure is of great significance for ensuring genomic stability.The helicase is a molecular motor protein that relies on the hydrolysis of ATP to gain energy to open hydrogen bonds between nucleic acids.Saccharomyces cerevisiae Pif1 is a kind of 5'-3'DNA helicase.Besides of resolving G4s,ScPif1 plays an important role in inhibiting telomerase activity,processing Okazaki fragment and replication of ribosomal DNA.Although the amount and distribution of G4 DNA in yeast has been detected by bioinformatics,the structural features of G4 DNA in Saccharomyces cerevisiae are still unknown,and the preference of Pif1 for different G4 structures is unclear.In this paper,we randomly selected 83 sequences of G4 in yeast,and systematically addressed the structural characteristics of G4 in yeast and its relationship with Pif1 by smFRET technique(single molecule fluorescence resonance energy transfer),CD spectroscopy and FRET Melting technology.We finally found the following results:(1)The sequence capable of forming the G4 structure is between 30 and 60 bases in length,and is more than 60 bases,it is difficult to form a G4 structure.And as the G4 stability decreases as the sequence becomes longer.(2)G4 structures can be formed with a broad range of loop sizes in vitro,the shortest loop length is 1 base,and the longest can reach 30 bases or more.Among the G4 structures,the parallel structure is favored.(3)Pif1 unfolds different G4 structures with different efficiencies.The thermal stability,configuration and loop length of G4 are important factors affecting the unwinding efficiency.Pif1 is easier to resolve anti-parallel G4 with longer loop and poor thermal stability.(4)Unwinding of the downstream duplex by Pif1 is limited by how fast G4 structure is unfolded,and G4 structure can either stimulate or impede the unwinding activity of Pif1.(5)Most of the G4 structures in yeast have longer loop and lower stability.Therefore,Pif1 can unfold majority of the G4 structure in yeast.However,Pif1 demonstrates poor activity towards G4 structures with short loops,so other helicases may be needed to resolve these G4 structures.The traditional calculation method generally considers that the length of the G4 loop is no more than 7 bases.This experiment first reported that the G4 sequence in the yeast genome can form G4 with a loop length of more than 30 bases in vitro,which greatly updated our understanding about G4 structures.In addition this study highlights the formidable capability of Pif1 to resolve the majority of G4s in S.cerevisiae sequences,narrows the fractions of G4s that may be challenging for genomic stability,and provides a framework for understanding the influence of different G4s on genomic stability via their processing by Pif1.