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Study On Structure And Biological Function Of Bacillus Subtilis RecQ Helicase

Posted on:2014-08-16Degree:MasterType:Thesis
Country:ChinaCandidate:Z R HuangFull Text:PDF
GTID:2180330482973157Subject:Biomedicine
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The RecQ helicase family which is highly conserved across a wide variety of organisms is an important member of SF2 superfamily of DNA helicase. RecQ helicases play essential roles in numerous metabolic pathways, such as DNA replication, repair, recombination, transcription, even telomere stability to maintain the stability of the genetic material. In this paper, we took E.coli RecQ and B.subtilis RecQ helicases as models, compared the dissimilarity between E.coli RecQ and two wild type isoforms of B.subtilis RecQ, carried on gene cloning, protein purification and determined the biochemical activity of E.coli RecQ, B.subtilis RecQ and its zinc finger mutants with biochemical and biophysical techniques. All the results are helpful to illustrate the relationship between protein structure and function. Following were the results:Firstly, The DNA corresponding to the coding sequence of the E.coli RecQ and B.subtilis RecQ helicases gene were amplified by PCR amplification,then amplified products were subcloned into the expression vector pET24a(+). The recombinant proteins were induced to express in Escherichia coli BL21(DE3) with IPTG at low temperature. After optimizing expression conditions, good solubility was obtained in vitro and helicases were with about 90% purity by nickel chelate affinity chromatography.Secondly, we analyzed the biochemical properties of two isoforms of Bacillus subtilis RecQ and E.coli RecQ. The three proteins were shown to have DNA-dependent ATPase activity in concentration-dependent manner, DNA binding activity, ATP-dependent DNA unwinding activity and time-dependent DNA annealing activity. B.subtilis RecQ S is naturally lack of HRDC domain. Our studies demonstrate that HRDC domain strengthened the DNA binding activity of Sub L while impaired its ATPase, unwinding and annealing activity.Subsequently, to explore the role of the zinc-binding motif, the pET24-B. subtilis RecQ S encompassing zinc finger motif was used as the target plasmid for site-directed mutagenesis. All point mutations were constructed using recombinant PCR, with the desired mutations were introduced in the internal mutagenic primers. Our first attempts to generate several single-point mutants C431G, C431R, C431A, C453R, C454S, C457G and double-point mutants C431G/C453R, C431G/C454S, C431G/C457G, C453N/C454N failed because these proteins were found to be rapidly degraded, which made it impossible to analyze their biological activity.These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein.In the last part, we generated a truncated variant of B. subtil is RecQ S comprised of conserved helicase domain only and its biochemical activity was significantly impaired compared with wild B.subtilis RecQ S.Based on the results presented above, we can conclude that HRDC domain is helpful in Bacillus subtilis RecQ helicase in DNA binding, but the enzyme without HRDC domain shows stronger ATPase activity, DNA unwinding and annealing activities than the other one. Besides, zinc-binding motif was proved to play an important role in DNA binding and protein folding.
Keywords/Search Tags:E. coli RecQ helicase, B.subtilis ReeQ S, B.subtilis RecQ L, protein expression and purification, zinc finger, HRDC domain, helicase domain, site-directed mutagenesis, biological activity
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