Sista P R, Hutchinson C A, Bastia D
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Genes Dev. 1991 Jan;5(1):74-82. doi: 10.1101/gad.5.1.74.
Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific DNA sequences (tau), constituting the replication terminus. Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites. With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis. The results show that one monomer of ter protein binds to a single tau site with an equilibrium dissociation constant of 5 x 10(-9) moles/liter. Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the tau core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo. The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a tau sequence. Thus, the asymmetric ter-tau complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a tau site.
要理解在序列特异性复制终点处DNA复制特异性且极化终止的分子机制，需要详细分析终止蛋白（ter）与构成复制终点的特定DNA序列（tau）之间的相互作用。此类分析应能为体内和体外在tau位点观察到的复制抑制功能极性提供结构基础。出于这一目的，我们已将大肠杆菌的复制终止蛋白纯化至同质，并使用化学探针和定点诱变技术分析了该蛋白与R6K复制终点的相互作用。结果表明，ter蛋白的一个单体以5×10⁻⁹摩尔/升的平衡解离常数与单个tau位点结合。此外，烷基化干扰与保护、羟基自由基足迹法和定点诱变相结合，揭示了tau核心序列中与ter蛋白接触的磷酸基团和碱基残基，以及那些对体内DNA-蛋白质相互作用和复制终止都很重要的残基。这些分析得出的总体情况表明，ter与tau序列形成不对称复合物。因此，不对称的ter-tau复合物为移动的复制叉在tau位点处停滞的功能极性提供了结构基础。
