Enzymatic Properties And DHBN Structure Investigations Of BLM Helicases

RecQ family helicases,named after the recQ gene of E.coli,are the essential genome guardians that almost function throughout the DNA metabolism processes,including DNA replication,repair,recombination,transcription and telomere maintenance.In human,there are five dominant RecQ helicases,mutations in three RecQ genes of blm,wrn and recq4 give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome(Bloom syndrome,BS),Werner syndrome(Werner syndrome,WS),and Rothmund-Thomson syndrome(Rothmund-Thomson syndrome,RTS),respectively.Among them,the cells of BS patients manifest the hallmark of about 10-fold increase in sister chromatid exchanges(Sister chromatid exchanges,SCEs)and are prone to a wide spectrum of malignant tumors.Since all characterized RecQ family helicases display very similar structural and functional similarities in helicase core domains,the significant differences in N-terminal domains might be essential for determining specific function of each helicase in cell.However,up to date,structure of the BLM N-terminal domain is still absent,and structure investigations on the whole BLM protein is also limited due to its low stability,low yield and high cost for large scale purification.Firstly,considering the above reasons,and based on comparisons of the sequences and expression systems of the BLM homologs,this study took Gallus gallus BLM helicase(gBLM)which shows a closer relationship with homo sapiens BLM helicase(h BLM)as a model,and developed the heterologous high-level expression and high-yield purification systems for gBLM and the truncated gBLM Core and gBLM C?HRDC in E.coli.This can provide abundant high quality proteins for further biochemical and biophysical studies.Then,by using gel-filtration chromatography analysis,dynamic light scattering(DLS),fluorescence anisotropy DNA binding assays and the stopped-flow DNA unwinding determination analysis,enzymatic property determination of gBLM obtained the following conclusions:(1)gBLM is a vigorous atypical DNA structure specific helicase which not only shows high preference for the 3'-tailed DNA structures but also can efficiently unwind the bubble DNA structures with blunt-ends.This indicates the important biological roles of gBLM in processing DNA metabolism intermediates.(2)Further comparative activity analysis among the different gBLM truncations reveal that the long N-terminal domain is not only involved in oligomerization of gBLM,but also can obviously facilitate binding to the forked and bubble DNA structures,and it is also required for the unwinding activities of the majority of DNA substrates.(3)Comparative activity analysis also shows that the HRDC domain plays a beneficial role in the binding and unwinding activities of gBLM with the most complicated DNA substrates.Thus,the high level and high purity purification strategy of gBLM and its enzymatic property characterization provide a new model for probing the molecular mechanism and structure of hBLM.Secondly,based on the above characterizations of gBLM,we further performed investigations on the BLM N-terminal domains by combination of bioinformatics,biochemical and structural approaches,and we obtained the following results:(1)Bioinformatics analysis over the sequenced 78 kinds of BLM homologous proteins showed that BLM N-terminal domains vary greatly within and across species,and the N-terminal domains are less conserved and poorly structured,it is unlikely that the N-terminal domains have conserved three dimensional structures.However,DHBN(Dimerization Helical Bundle in N-terminal domain)which contains potentially 2-3 alpha helices is the only highly conserved domain in the N-terminal domains of the vertebrate BLM proteins,implying its important biological role throughout the evolution process.High structure stability of gDHBN was also evidenced by the limited proteolysis and LC-MS/MS identification results of the different gBLM N-terminal truncate proteins.(2)Through systematically crystal screening,optimization and X-ray crystallography for the different BLM N-terminal truncates,we determined high resolution crystal structures of hDHBN(2.0 ?),gDHBN(2.7?)and pDHBN(1.4?)from hBLM,gBLM and pBLM(Pelecanus crispus BLM helicase),respectively.Further interaction analysis and biochemical characterization reveal that the highly conserved DHBN domain is stabilized largely by virtue of hydrophobic interactions and is mainly involved in dimerization of the BLM helicases.The SAXS determination model of gDHBN in solution also confirmed this dimeric structure.(3)Gel-filtration chromatography analysis and DLS measurements of the different gBLM truncated proteins show that the conserved dimeric DHBN is the basic unit for BLM high order oligomer assembly.Based on these structures,we propose the oligomeric ring structure models of hBLM observed in the previous electron microscope experiment.Additionally,the DNA unwinding activity assay and DLS determination comparisons of the gBLM truncate proteins also reveal that the DNA unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer,suggesting a regulative role of DHBN in the DNA unwinding process.Interestingly,the stable DHBN dimer can be dissociated upon ATP hydrolysis in the context of the full-length BLM protein,which was consistent with dissociation characteristics of hBLM and WRN under ATP conditions.Therefore,these biochemical and structural characterizations not only firstly establish a high purity and high level purification strategy,reveal the regulative roles of its N-terminal domain on oligomerization and enzymatic activity of gBLM helicase,but also firstly reveal the high sequence-structure conservation of BLM DHBN domain and its potential regulative roles.These findings will not only deepen our understanding on the enzymatic properties and structure similarities of the BLM homologues,but will also provide new evidences for the structural and functional organization of hBLM.