Multiple Regulatory And Mechanisms Of RPA On The Formation And Unwinding Of G4-stranded DNA

Replication protein A(Replication protein A;RPA)is the main single-stranded DNA(ss DNA)binding protein in eukaryotic cells.It is a highly flexible modular protein and is regarded as the first responder to act on the exposed ss DNA.RPA mainly plays a role in protecting the single strand from being degraded by other nucleic acid proteases and preventing the formation of secondary structure of the single strand in the cell.RPA provides a platform for other proteins to enter the site of action through interaction with other proteins.RPA also acts as a sensor that senses genomic damage caused by multiple sources.The G-quadruplex(G4)structure is a four-stranded helical structure formed by the folding of a continuous guanine-rich DNA sequence.Once the G4 structure is formed,it has high thermal stability.G4 is usually regarded as an obstacle in the replication,transcription and repair of genes.In order to quickly remove the G4 DNA structure,helicase or other proteins are required to open it,otherwise it will affect gene metabolism.Based on genome sequencing results,there are over 700,000 potential G4 folding sites in the organism.RPA can open some G4 structures in the process of protecting single chain and recruiting other proteins for interaction.The ability of RPA to guide DNA processing depends to a large extent on the interaction of RPA with other proteins in various processing pathways.Almost all DNA processing pathways in the cell require RPA participation,such as the G4 metabolism process involved in RPA.It has been found that RPA can inhibit the formation of G4 on the leading and trailing chains.RPA mutations lead to a large amount of G4 accumulation in cells.It has also been found that RPA can work with other helicases to eliminate the leading and trailing chain G4 structure.However,these results are currently observed at the cellular level,and their specific mechanism is unclear.Our study uses biochemical methods to explain the mechanism of RPA inhibiting the formation of G4 in vitro,which has certain physiological significance.During DNA replication,transcription,and repair,if the transiently exposed ss DNA contains G4 folding sites,it is still unclear whether it actually folds into a G4 structure in vivo.Once the G4 structure is formed,can RPA open it? It is unclear whether RPA has a preference for different G4 structures and what its unwinding mechanism is.It has been reported in the literature that RPA has the function of recruiting other proteins.At the single molecule level,how RPA interacts with other helicases to unwind G4 substrates is still unknown.In this study,single-molecule fluorescence resonance energy transfer technology was used,combined with circular dichroism chromatography and FRET melting experiments to study the interaction between RPA and G4 DNA.This experiment has the following conclusions: First,RPA interferes with the formation of G4 structure.The potential G4 sequence is not necessarily folded into a structure when it is exposed temporarily.It mainly depends on the difference between the internal folding rate of G4 sequence and the rate of RPA binding to ss DNA,and is closely related to the stability of G4,sequence length,and RPA concentration.Second,for the folded G4 structure,RPA expands it selectively with different behaviors.We propose a mechanism for G4 DNA respiration,and classify the untwisting behavior of RPA into three categories: a.Continue to open low-stability G4 and open single strands stably with multiple RPA molecules;b.Open-fold quickly The G4 is opened with high stability,but G4 with spontaneous breathing,including Myc G4;c,RPA has almost no destructive effect on multi-layer G4 and long Loop G4 without breathing.Third,RPA cooperates with Pif1 or BLM helicase to produce a new phenomenon when unwinding G4,stabilizing the structure of G4 in a state of continuous expansion,and greatly improving the unwinding efficiency of G4 structure.This paper reveals that RPA has multiple functions in the folding and opening of G4 DNA,which is significance for the in-depth understanding of the microscopic regulatory mechanism of G4 structure in the process of DNA replication,transcription,repair and telomere metabolism.