大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族，在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达，并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶，其可溶性好；经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA），大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 )，但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05)；大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05)；大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制，并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.

Analysis of Biological Activity of Escherichia coli RecQ Helicase

The RecQ family helicases belong to an important family of highly conserved DNA helicases that play a crucial role in the maintenance of chromosomal stability, and their mutations have been led to several disorders and cancers in human. The study is aimed to induce the expression in vitro of E. coli RecQ helicase, and to determine its biological activity using biochemical and biophysical techniques. The E. coli RecQ helicase with above 90% purity and activity was obtained in vitro, and good solubility. The E.coli RecQ helicase was shown to have DNA-binding activity, ATP-dependent DNA unwinding activity, and DNA-dependent ATPase activity. The E. coli RecQ helicase was easier to bind with single-stranded DNA (ssDNA) than double-stranded DNA (dsDNA) (P<0.01) with the difference existing in varying lengths of dsDNA(P<0.01) but not the ssDNA (P>0.05). There was a difference in DNA unwinding rate of the helicase for three lengths of dsDNA (P<0.05), and a positive correlation existed between the ATPase activity of the helicase and the length of ssDNA cofactors (P<0.01). These results are helpful to elucidate the molecular mechanisms of the E. coli RecQ helicase and study the structure and function of other members of the RecQ family helicases.