摘要：
Mycobacterium tuberculosis is the pathogen of tuberculosis which causes about millions people death annually.M.smegmatis,as a type of non-pathogenic strain of Mycobacterium closely related to M.tuberculosis,is the most studied model strain in laboratory.Mycobacterium encodes three types of chromatin proteins,i.e.histone-like protein HU,Lsr2 and integration host factor (IHF).To investigate the functional roles of IHF in chromosomal DNA organization,we expressed and purified IHF protein from M.smegmatis (MsIHF) in Escherichia coli,and analyzed the effects of MsIHF on DNA topology in vitro.MsIHF exists as a stable homodimer in solution.MsIHF exhibits preferred binding to negatively supercoiled DNA rather than linear or relaxed DNA.This protein is also capable of constraining negative DNA supercoils.Further analyses showed modulations of topoisomerase activities by MsIHF in vitro.MsIHF obviously inhibits the relaxation of supercoiled DNA by topoisomerase Ⅰ from E.coli.By contrast,this protein slightly stimulates E.coli gyrase to introduce negative supercoils into relaxed DNA.These data suggests that MsIHF may alter the topological structure of chromosomal DNA through modulation of the activities oftopoisomerases,and therefore regulates the organization of chromosome.%结核分枝杆菌(Mycobaterium tuberculosis)是结核病的病原菌,每年导致数百万人死亡.对于分枝杆菌基本生物学特性的研究有助于新的药物及治疗手段的研发.耻垢分枝杆菌(M.smegmatis)是分枝杆菌属中的一种非致病菌,与结核分枝杆菌亲缘关系较近,是实验室常用的研究分枝杆菌的模式菌种.分枝杆菌主要编码三种染色质蛋白,类组蛋白HU、Lsr2和宿主整合因子IHF.为研究IHF在染色体包装中的作用,我们在大肠杆菌中表达、纯化了耻垢分枝杆菌IHF蛋白(MsIHF),并对其影响DNA拓扑结构的性质进行了系统分析.体外研究的结果表明,MsIHF以同二聚体的形式存在,其对负超螺旋DNA具有一定的结合偏好性,同时,该蛋白可以有效地固定DNA负超螺旋.进一步的研究表明,MsIHF可以调控拓扑异构酶的活性.MsIHF的结合明显地抑制拓扑异构酶Ⅰ的松弛活性,而与此相反,该蛋白可以轻微地促进旋转酶引入DNA负超螺旋的能力.以上结果提示,MsIHF可能通过调控拓扑异构酶的活性影响染色体DNA的结构,进而调控其包装.
