The Study Of Small Molecules Effect On The Activities Of RecQ Helicase Family Members

Helicase can utilize the energy released from the process of nucleoside triphosphate hydrolysis in order to translate along the nucleic acid strand, and then separate one double-stranded DNA into two singled-stranded DNA. As a representative of the helicases, RecQ helicase family is an important protein factor which can play a significant role in maintaining the stability of chromosome genome and telomere. More importantly, the genetic mutations among the family members in human(BLM, WRN and RECQ4) will lead to three kinds of serious diseases which are named as Bloom, Werner and Rothmund-Thomson syndrome respectively, and these diseases are always accompanied by a high risk of cancer occurrence. In addition, another RecQ family member, RECQL helicase, may be associated with the breast cancer and pancreatic cancer.When helicases perform a physiological role in cells, some kind of small molecules may regulate or influence the helicase activity. For example, adenosine triphosphate(ATP) can regulate the oligomer states of protein, thus affecting its unwinding activity towards DNA substrates. Moreover, some heavy metal compounds may also have potential impact on the activities of helicase. For instance, cadmium chloride(CdCl2) intake or BLM-deficiency in cells will result in the same cytological phenomenon that the increasing of sister chromatid exchanges, which suggests the connection between cadmium and BLM. Therefore, this experiment selected two groups of subjects, RECQL/ATP and BLM/CdCl2, in order to clarify the impact of small molecules on the activities of helicases. Then, the dependency between protein and compound was analyzed in detail and the changes of helicase activities were also determined. First of all, the prokaryotic expression system was carried out together with different purification technology(affinity, ion exchange or gel filtration chromatography) to obtain the protein sample with high purity. Subsequently, we conducted the dynamic light scattering, fluorescence anisotropy and stopped-flow spectroscopy assays to study the effect of small molecules on the activities of helicase including multimer states, DNA binding and unwinding activities. All in all, we hope to get a deeper and comprehensive understanding on helicases.The experimental results of ATP affecting RECQL activities indicated that(1) RECQL usually existed as a tetramer formation and multimers were immediately dissociated into stable dimers upon ATP binding. In other words, ATP was the switch controlling the assemble states of RECQL. Besides, the RECQL mutants, RECQL49-616 and RECQLWH-AAA, were a single dimer and monomer respectively. However, ATP couldn’t change the oligomer states of these two mutants;(2) The distinct oligomer states of proteins were accompanied with a different DNA binding affinity. Specifically, fluorescence anisotropy assays revealed that the favoured DNA substrates for binding were blunt-dsDNA for RECQL and ssDNA for RECQL49-616. Moreover, there was an obvious dependency between binding affinity and the presence of 3’-tailed ssDNA in G4 DNA structure when RECQL bonded with G4 DNA substrate;(3) ATP could affect the different oligomeric formation of RECQL, thus regulating the varying activities of helicase, which tetramer and dimer were responsible for DNA strand annealing and unwinding respectively;(4) We carried out the multiple-turnover unwinding kinetics with DNA substrates of different 3’-tail ssDNA lengths, steady-state unwinding kinetics and varying ATP concentrations unwinding assays in order to determine the functional oligomer state of RECQL in resolving DNA structures. More importantly, it was proved that RECQL unwound duplex DNA in a single and stable dimeric form;(5) The different assemble states of proteins preferred distinct DNA substrates for unwinding. Surprisingly, RECQL was capable of unwinding Holliday junction, but RECQL49-616 unwound duplex DNA in a manner that was much higher efficiency than RECQL and RECQLWH-AAA;(6) For the first time, we found that RECQL was able to make G4 DNA structure dissociate into a ordinary ssDNA and RECQL49-616 could weakly unwind Holliday junction under our experimental conditions. Collectively, our study successfully took advantage of Escherichia coli expression system combined with protein purification techniques in order to obtain Bos taurus RECQL helicase with remarkable quality at first time. Moreover, the stopped-flow spectroscopy assays were used to analyze the activities of RECQL for the first time. Altogether, it was indicated that ATP binding could regulate the multimer states of RECQL in order to accomplish the different activities and the dimer of RECQL was in charge of the unwinding process. These findings would be meaningful evidence for further research on the working mechanism of RECQL helicase.The experimental results of cadmium affecting BLM activities suggested that(1) Cadmium could significantly inhibit both BLM unwinding activity and ATPase activity;(2) There were two kinds of different cysteine residue groups on BLM with distinct affinity to cadmium;(3) Cadmium strongly prevented the DNA binding activity of BLM through interacting with sulfhydryl groups of cysteine residues located in protein surface;(4) It was found that zinc finger domain of BLM appeared to play a minor role during the inhibition of BLM-DNA binding activity by cadmium, which was quite different from the existing reports;(5) Cadmium could promote BLM monomers to form the larger BLM aggregates and this effect was fully counteracted by EDTA;(6) Dithiothreitol was required for the inhibition of BLM binding activity by cadmium. Meanwhile, the varying DTT concentrations would cause different influence on the inhibition processes. Moreover, the defect of binding activity was reversible upon addition of EDTA as well;(7) The inhibition of BLM-DNA unwinding activity by cadmium was not recovered by EDTA. All in all, our studies provided new clues for understanding the mechanism behind the zinc binding domain independent inactivation of BLM by cadmium. Besides, these results also gave important information about the role of BLM playing in the process of homologous recombination repair and the stability of genome maintaining.