Patel S S, Hingorani M M
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Biophys J. 1995 Apr;68(4 Suppl):186S-189S; discussion 189S-190S.
Bacteriophage T7 DNA helicase protein is a hexameric protein that contains identical subunits arranged in a ring-like structure. Single-stranded DNA binds through the hole of the ring, and the helicase protein translocates and unwinds duplex DNA using nucleoside triphosphate (NTP) hydrolysis. In our efforts to understand how NTP hydrolysis may be coupled to movement of the helicase on the DNA, we have quantitated the equilibrium binding of deoxythymidine triphosphate and thymidine 5'-(beta,gamma-methylenetriphosphate) using nitrocellulose binding assays. Even though the helicase consists of six identical subunits, each hexamer was found to bind only three NTP molecules. These results indicate half-site binding or negative cooperativity in NTP binding by the hexamer. Interestingly, binding of three NTP molecules to the hexamer was sufficient for stoichiometric binding of a single-stranded oligodeoxynucleotide. Similar negative cooperativity in NTP binding has also been observed for other helicases, suggesting that it may be a general feature of hexameric helicases. The significance of half-site binding, however, is not understood at the present time.
噬菌体T7 DNA解旋酶蛋白是一种六聚体蛋白，由排列成环状结构的相同亚基组成。单链DNA通过环的孔结合，解旋酶蛋白利用核苷三磷酸（NTP）水解进行转位并解开双链DNA。在我们试图了解NTP水解如何与解旋酶在DNA上的移动偶联的过程中，我们使用硝酸纤维素结合试验对脱氧胸苷三磷酸和胸苷5'-（β，γ-亚甲基三磷酸）的平衡结合进行了定量。尽管解旋酶由六个相同的亚基组成，但发现每个六聚体仅结合三个NTP分子。这些结果表明六聚体在NTP结合中存在半位点结合或负协同性。有趣的是，三个NTP分子与六聚体的结合足以实现单链寡脱氧核苷酸的化学计量结合。在其他解旋酶中也观察到了类似的NTP结合负协同性，这表明它可能是六聚体解旋酶的一个普遍特征。然而，目前尚不清楚半位点结合的意义。
