Enzyme Properties And Structures Study Of DNA Helicase CtRecQ And RNA Helicase DDX21 In SF2

Superfamily 2（SF2）is a large and structurally diverse superfamily of helicases.The majority of the proteins in SF2 are primarily involved in DNA repair,chromosome segregation,and transcription control,among other things.This superfamily helicase also possesses a number of linked auxiliary domains that help the unwinding core domain carry out numerous tasks,including chromatin modification and the production of synthetic peptides.The RecQ family helicases carry out a variety of DNA metabolic pathways crucial for preserving genomic integrity,and they have undergone significant evolutionarily conserved adaptation in SF2.Aberrant RecQ helicase expression results in high genomic instability,chromosomal abnormalities,DNA damage sensitivity,and other diseases.Due to the in vivo and in vitro characteristics of the protein,the structural study on the human RECQL4 and yeast Hrq1 homologues is somewhat behind.In an effort to better understand the process of nucleic acid metabolism,we produced the Chaetomium thermophilum RecQ helicase protein and examined its structure,function,and mode of action.reveal the mechanism underlying some malignancies and offer fresh targets and concepts for the creation of anticancer medications.The prokaryotic expression system,along with dynamic laser scattering,rapid stop-flow,fluorescence resonance energy transfer,and X-ray crystallography diffraction technologies,were used in this study to examine the enzymatic properties of CtRecQ helicase.The results are listed below.According to research on sequence preference of CtRecQ^121-1198 and ability to bind substrates,the ability to bind ss DNA and ds DNA blunt-end substrates increased with single-strand length.The binding ability of substrates with 3’tail strands is superior to substrates with 5’tail strands for ds DNA overhangs,and the longer the tail strands,the greater the binding ability.The base sequences d T and d G are preferred by CtRecQ^121-1198for binding.The length of the tail chain must be longer than 30 nt for CtRecQ^121-1198 to exhibit unwinding activity since it is a stringent 3’–5’helicase.The unwinding action is unaffected by the N-terminus,and the C-terminal domain is essential for it.CtRecQ^121-366 is a 3’direction ss DNA and ds DNA exonuclease that is independent of the short-chain ss DNA sequence and has a lower degradation efficiency for ds DNA than for ss DNA.The 3D structure of CtRecQ^121-366 was modeled utilizing afold2.Similar to most exonucleases,has a three-dimensional structural conformation.7 helices and 3 sheets make up the entire structure.The exonucleases’ability to function requires helix 6 and 7.CtRecQ^369-1198 is divided into three sections for its general structure.A"triangular"configuration is formed by the two helicase core domains and the C-terminal accessory domain.WH,OB,CON,DUF1998,Rec A1,and Rec A2 domains are present.Only the Mrf A protein possesses the special U-motif.Rec A1 and Rec A2 are joined by the U-motif through ten pairs of hydrogen bonds,which is crucial for preserving CtRecQ general conformation.These studies add to our knowledge of RecQ helicase’s unwinding process and contribute to our understanding of the specific function of RecQ helicase in preserving genome stability.The DEAD-box RNA helicase DDX21 catalyzes the unwinding of structured RNA,the dissociation of RNA complexes,and the remodeling of RNA-protein complexes using the energy produced by ATP hydrolysis in SF2.RNA polymerase II（RNA Pol II）-mediated transcription and ribosomal RNA processing are two of the several biological activities in which DDX21 is active.DDX21 is attracted to the promoters of RNA Pol II-transcribed genes generating ribosomal proteins and small nuclear RNAs（sno RNAs）in the nucleoplasm after binding to 7SK RNA inside the 7SK nucleosmall ribonucleoprotein particle（sn RNP）complex.Changes in DDX21 have been linked to a number of illnesses,such as breast cancer,stomach cancer,and melanoma.DDX21 is a dual-purpose enzyme.Research into the DDX21 bifunctional enzyme’s structure,function,and mechanism of action can not only shed light on the nucleic acid metabolism process but also disclose the mechanism behind various malignancies and offer new targets and concepts for the creation of anticancer medications.This study examines the enzymatic properties and DNA annealing activity of the helicase DDX21 using a variety of research approaches,including protein purification and separation technology,fast residence technology,biological macromolecular crystallography technology.The annealing model was clarified,and the following primary study findings were attained:1)On the basis of bioinformatics prediction and structural data,human DDX21truncated proteins of various lengths were produced in vitro,and high-purity and high-quality full-length and truncated proteins were obtained.Using fluorescence anisotropy to detect binding activity.The findings demonstrated that DDX21 was more able to bind RNA than DNA substrate.The length enhances the binding capacity of the same kind of substrate.DDX21^564-783 has a greater binding activity than the full-length protein.2)DDX21 unwinds ds RNA but not ds DNA.DDX21 has the strongest ability to unwind Fork-ds RNA type substrates Am=57.9%,which is twice as strong as unidirectional tail strand substrates,for ds RNA substrates.Unwinding activity can be produced by the unwinding core on its own,and the C-terminal accessory domain serves to increase unwinding activity.3)Both DNA and RNA can be annealed by DDX21,however DNA substrates exhibit stronger annealing activity than RNA.The DNA substrate annealing activity of the DDX21C-terminus（564–783）is identical to that of the full-length protein.The DD,GUCT,and Repeats domains at the C-terminus all have a significant impact on annealing activity.The DD domain increases the annealing rate,while the GUCT and Repeats domains are essential critical domains for annealing activity.The N-terminal,DD,and unwinding core domains control the majority of the full-length protein’s annealing activity.4)The full-length protein of DDX21 preferentially binds to the 3’end of the DNA substrate,attracting complementary DNA strands to complete annealing,and without requiring ATP,according to the analysis of annealing activity based on Stop-flow.5)After truncating the entire protein and putting it through a protease digestion,the conserved domain structure DEADc of DDX21^178-398 was determined.In conclusion,the SF2 family member DNA helicase CtRecQ depends on the N-terminal nuclease activity to expand the helicase activity in order to finish double-stranded DNA unwinding and degradation;the RNA helicase DDX21 works in conjunction with the helicase activity through the C-terminal annealing activity or competes for nucleic acid substrates in order to perform a diversity function.