Using Fluorescence Technology To Study The Interaction Between Ding And RecD2 Helicase And DNA

Due to the complexity of biological reactions,traditional bulk detection techniques can detect the overall changes in the reaction process,but it is difficult to visually detect the molecular processes and transient states of biological reactions.In recent years,single-molecule technologies including single-molecule fluorescence resonance energy transfer,magnetic tweezers,etc.,which have emerged and become mature,have improved the ability of people to study the behavior and mechanism of biological macromolecules in real time;this article uses bulk and single-molecule fluorescence technology to study the interaction between protein and DNA.The process and mechanism of action,we can fully understand the process of biological reactions,greatly enhancing people's ability to understand complex life processes.The Din G protein(Ec Din G)of Escherichia coli containing iron-sulfur clusters can quench Cy3 fluorescent dyes,but the specific mechanism is not clear.The possibility of electron transfer between iron-sulfur clusters and Cy3 was investigated by adding oxidant hydrogen peroxide and reducing agent DTT,which proved that Cy3 quenching can be used as a specific and sensitive probe for studying the specific movement of helicase and DNA.Nucleic acid is a carrier of genetic information,composed of deoxyribonucleic acid(DNA)and ribonucleic acid(RNA).It is known that the life activities of all organisms cannot be separated from a series of reactions such as nucleic acid transcription,replication,and translation,and this requires Helicase opens the hydrogen bonds between nucleic acids.Once there is a problem in the unwinding process,the organism will have disease and cannot survive normally.Therefore,it is particularly important to study and understand the unwinding behavior and potential mechanisms of different nucleic acid structures by helicase.When the cell is disturbed by external conditions,the nucleic acid structure is easily destroyed.Deinococcus radiodurans has a strong ability to resist DNA damage and has been used as a model for studying DNA repair mechanisms.It does not have Rec BCD(helicase)or Add AB(nuclease)which is essential for the repair of bacterial double-stranded DNA breaks,but it does have DrRecD2,a homologue of Rec D,the Rec BCD helicase unit.DrRecD2 helicase,as a structural model for studying superfamily 1B(SF1B)helicases,plays an important role in the repair of DNA strand breaks in Deinococcus radiodurans,but the specific behavior and mechanism of DrRecD2 unwinding double-stranded DNA is still unknown..Although double-stranded DNA accounts for a large proportion in organisms,DNA can be transformed into other structures.G4-quadruplex DNA is a common secondary structure in Deinococcus radiodurans genome,which plays an important role in the life activities of cells,but sometimes the G4 structure can become an obstacle to the DNA replication mechanism.It is necessary to untie the G4 structure DNA.In order to complete normal physiological activities.Unfortunately,the G4 helicase in Deinococcus radiodurans has not been well identified.Therefore,the study of the unwinding mechanism of DrRecD2 unwinding G4-quadruplex DNA is essential for maintaining genome integrity and stability during replication.very important.In this study,the iron-sulfur cluster protein was firstly reacted with Cy3-labeled single-stranded DNA with different chain lengths and hydrogen peroxide and DTT were added.Through the measurement of Cy3 fluorescence intensity and bulk life,it can be known that the shorter the DNA chain length,the Cy3 The more severe the fluorescence quenching is;during the reaction between the iron-sulfur cluster and Cy3,Cy3 transfers electrons to the iron-sulfur cluster.Then,through single-molecule technology operations(total internal reflection fluorescence microscope and magnetic tweezers),the mechanism of action of DrRecD2 helicase to unwind double-stranded and G4-quadruplex DNA was explored.By labeling the fluorescent dyes Cy3 and Cy5 on single-stranded DNA and G4 structured DNA with different chain lengths,and then heating and cooling the complementary single-strands on a PCR machine for annealing and recombination,the single-stranded DNA is connected to double-stranded by hydrogen bonding.In the fluorescence resonance energy transfer experiment,streptavidin was used to connect the biotinylated DNA in a PEGylated glass reaction chamber,and then different concentrations of DrRecD2 helicase,ATP(to provide energy),search DNA focal plane for data collection and analysis.Use tools such as a single-molecule total internal reflection microscope system and magnetic tweezers to reflect the changes in the fluorescence intensity of the donor Cy3 and the acceptor Cy5 during the process of fluorescence resonance energy,and infer the change in the distance between the donor Cy3 and the acceptor Cy5 to obtain the internal structure of the DNA change.The following conclusions can be drawn through research:(1)DrRecD2 unwinds the shorter double-stranded DNA(containing 17 base pairs)showing a one-step unwinding phenomenon.(2)DrRecD2 unwinds longer double-stranded DNA(containing 29 base pairs).Compared with 17 base pair DNA,the unwinding ratio and speed are significantly reduced and repeated unwinding occurs.The phenomenon of repeated unwinding is accomplished by a single helicase molecule,not by multiple helicase monomers.(3)The model of DrRecD2 unwinding longer double-stranded DNA is: when DrRecD2 reaches the unwinding limit,it will reach the complementary single-stranded DNA through strand switching,then shift backward,and finally switch and re-grasp the original strand.(4)DrRecD2 unwinding limit is affected by ATP concentration.The higher the ATP concentration,the longer the length of DrRecD2 unwinding double-stranded DNA.(5)DrRecD2 unwinding G4-quadruplex DNA affects its unwinding double-stranded part,G4 structure hinders the unwinding of DrRecD2.DrRecD2 can unwind parallel and antiparallel G4 structure DNA without obvious topological preference;while Sc Pif1 has stronger ability to unwind antiparallel G4 structure DNA.Therefore,this study provides a new function of DrRecD2 helicase,and may help to understand the mechanism and ability of the superfamily 1B(SF1B)helicase to unwind the DNA structure.