Study On Self-assembly Behavior,Enzymatic Characteristics And Mechanism Of RECQL Helicase

DNA helicase plays an important role in DNA replication,transcription regulation and repair.The RecQ family helicases are a family of ATP-dependent helicases,which play a central role in maintaining genome stability.Three defects in the five human RecQ homologs can lead to unique genetic diseases related to genome instability,cancer susceptibility and premature aging.Different RecQ family helicases have non-overlapping functions,but the molecular basis of their different enzymatic activities is still unclear.In addition,members of the helicase family have been shown to be able to solve atypical DNA structures,such as fork DNA,G quadruplex,D loop and Holliday Junction(HJ)structure.However,the understanding of the mechanism by which RecQ family helicases recognize and process their physiological substrates is still very limited.Helicase RECQL is the first human RecQ family helicase to be discovered.It has the largest amount in the body and the shortest structure.Its ATPase domain shared by all superfamily 2(SF2)helicases and the specific C-terminal domain of RecQ(RQC domain),and shorter N-and C-terminal extension structures.Cell pathway studies have shown that RECQ1 plays an important role in response to TOP1 inhibition,replication fork initiation process,and regulation of cancer cell migration and invasion.Research reports that RECQ1 is related to breast cancer disease.In vitro recombinant biochemical studies have shown that the assembly state of RECQ1 plays a crucial role in regulating its helicase,branched chain migration(BM)or chain annealing.In this paper,we combined with various research techniques in vivo and in vitro,such as molecular sieve separation technology,Western blot technology,fast stop technology,single molecule fluorescence resonance energy transfer technology,biological macromolecular crystallography technology,etc.,to study the helicase RECQL.The enzymatic properties of RECQL,the self-assembly behavior in vivo and in vitro,and its mechanism of action has been clarified from the structural level.The main research results shown as follows:First,the target protein BtRECQL helicase was overexpressed in Escherichia coli using in vitro recombination technology.The results of molecular sieve and dynamic laser light scattering technology showed that the helicase BtRECQL is tetramer or tetramer and dimer in solution.In the mixture,the combination of ATP/d ATP can promote the depolymerization of the helicase BtRECQL from the tetramer to the dimer;the results of rapid dwell isokinetic technology test show that the dimer is responsible for the unwinding activity,and the tetramer does not play the unwinding effect.,And has the effect of annealing complementary single-stranded DNA.And clarify that the regulator between the two activities of unwinding and annealing is ATP or d ATP;Secondly,starting from the endogenous helicase BtRECQL,combined with molecular sieve and Western bolt technology,it was found that the helicase BtRECQL in the total protein extract of bovine cecal tissue exists in two kinds of dimers and monomers,and the monomer content is high.For the dimer,no tetramer was detected.This result is completely different from the results of in vitro recombinant protein;in order to obtain a large amount of high-purity BtRECQL monomer,its function has been studied.The monomer BtRECQL was constructed in vitro,and its activity was studied.It was found that the speed of unwinding double-stranded DNA of monomer BtRECQL is tetramerized and dimerized.7-10 times of polymerization,the processing speed of Holliday Junction substrate is 3-6times of tetramerization and dimerization,and the speed of unwinding G4 DNA is more than twice that of tetramerization and dimerization.BLM helicase is equivalent;it shows that BtRECQL plays an important function in the body as a monomer.It is speculated that mammalian cells use an as yet undiscovered mechanism to maintain a certain amount of BtRECQL as a monomer to perform special functions.Thirdly,using the method of real-time monitoring of the unwinding of intramolecular G quadruplexes developed in this article,it is found that BtRECQL has a higher activity of unwinding telomere G4(Tel G4),and its unwinding amplitude is as high as 80%.BtRECQL unwinding parallel and anti-parallel G4 have the same unwinding amplitude,but the unwinding speed of the unwinding parallel structure G4 DNA is 7 times that of the anti-parallel structure;this result is the first report that RECQL helicase has unwinding G4 activity,this study Solved the long-standing puzzle: Among the members of the RecQ family whose primary sequence is conserved,why RECQL alone cannot open the G4 structure.Finally,through X-ray diffraction crystal structure analysis,it is found that,compared with dimers,BtRECQL monomer has fewer amino acids interacting with ADP-Mg,the monomer structure is more compact,and the ability to bind and utilize ATP is enhanced,leading to the increase of BtRECQL monomer.The unwinding activity is significantly enhanced;in addition,the analysis of the existing structure results found that the interaction of the dimer dimerization interface will make the RECQL monomers restrain each other,making it impossible to shift along the 3'-5' direction.It may be the main reason for the obvious decrease of dimer activity.This result expands on existing reports and suggests that the monomeric form may perform previously unrecognized functions in cells.