Enzymology And Crystallization Of RecQ Helicases WRN And RecQ1

RecQ family helicase have been highly conserved in the whole evolution process.Their functions involve many kinds of DNA metabolic processes and play essential roles in maintaining genome stability.In human,there are five RecQ homologs,and deficiencies in three of them,BLM,WRN and RecQ4,give rise,respectively,to Bloom,Werner and Rothmund-Thomson syndromes that are characterized by genomic instability,chromosomal abnormalities,and sensitivity to DNA damage at the molecular level.During the past decades,characterization of RecQ family helicases primarily based on purified recombinant proteins has demonstrated that RecQ helicases adopt different quaternary forms,including higher-order oligomers?likely pentamers or hexamers?and dimers.Furthermore,members of this helicase family not only unwind duplex DNA,but also efficiently process non-canonical DNA structures,such as forked DNA,G-quadruplexes?G4?,D-loops,and Holliday junction?HJ?structures.Therefore,the study on the structure,function and mechanism of RecQ helicase can not only elucidate the nucleic acid metabolism process,but also reveal the mechanism of some cancers,providing new targets and ideas for the development of anticancer drugs.WRN and RecQ1 are the two most representative RecQ helicases.RecQ1 is the first RecQ helicase found in humans.In addition to the helicase domain,WRN also includes the exonuclease domain.In this paper,WRN and RecQ1 were used as the research objects.The high purity WRN and RecQ1 protein samples were obtained from Escherichia coli prokaryotic expression system and purified by affinity,ion exchange or gel filtration chromatography.The enzymatic properties of RecQ helicase were studied by dynamic light scattering,stop-flow fluorescence detection,fluorescence resonance energy transfer technique and X-ray crystallography diffraction technique.The main achievements are as follows:Firstly,based on bioinformatics analysis,different length truncation of Gallus gallus WRN?gWRN?helicase was constructed and the conditions of expression and purification were optimized.It was found that removing the irregular gWRN Link?1033 aa-1132 aa?could effectively avoid protein degradation,while retaining the HTH domain of gWRN-C terminal could significantly improve the solubility and stability of protein,thus the core fragment gWRN^512-1338?l was obtained.Gel filtration and DLS studies on the oligomers state of gWRN with different truncated lengths showed that gWRN retained in the form of higher-order oligomers and hexamers when the N exonuclease domain was retained,while the helicase region was in the presence of hexamers,trimers,or monomers.Secondly,the analysis of gWRN helicase activity by Stop-flow fluorescence detection showed that gWRN helicase had strict 3'?5'unwinding polarity,and had different unwinding efficiency for different types of DNA substrates,especially efficiently unwound a forked duplex substrate.In addition,HRDC domain and HTH domain of gWRN helicase were not necessary in the process of DNA double strand unwinding.At the same time,progress has been made in the crystals screening of truncated gWRN helicase of different lengths.The gWRN^709-1338?l and gWRN^490-1338?lobtained protein crystals under the screening conditions.Through the optimization of crystallization conditions,gWRN^490-1338?lobtained a diffraction resolution of 9?.In addition,the monomer mutant BtRecQ1^mon of Bos taurus RecQ1?BtRecQ1?was constructed by mutating the RecQ1 dimer interaction site,and a high-purity protein sample was obtained.BtRecQ1^mon helicase kinetics assays show that monomers are more active than dimers and tetramers in DNA unwinding.The Rate of BtRecQ1 were dependent on the tail length of substrate,while Rate of of BtRecQ1^mon were independent on the tail length of substrate,indicated that BtRecQ1^mon catalyzes DNA unwinding as monomer at saturating enzyme concentrations.Through crystal structure analysis,it is found that compared with dimers,BtRecQ1 monomer has a denser structure,and its ability to bind and utilize ATP is enhanced,resulting in a significant increase in the unwinding activity of BtRecQ1^mon monomer.The BtRecQ1^mon monomer plays an important role in the unwinding of G4 DNA,and the?-hairpin plays a key role in the unwinding of G4 within the molecule of BtRecQ1.To sum up,this paper systematically carried out the expression,purification,and crystal screening of different length truncation of gWRN helicase,constructed the high-efficiency expression and purification system of WRN helicase,studied the enzymatic characteristics of gWRN unwinding,and obtained gWRN protein crystal with certain diffraction ability,which laid the foundation for the analysis of WRN three-dimensional structure.At the same time,the study on the structure of BtRecQ1 and the kinetics of unwinding showed that the monomer structure was a functional oligomeric state in the process of ds DNA and G4 DNA unwinding mediated by BtRecQ1 helicase.These studies expand the current understanding of the mechanism of RecQ helicase,and help to further understand the specific role of RecQ helicase in maintaining genomic stability.