Substrate requirements for duplex DNA translocation by the eukaryal and archaeal minichromosome maintenance helicases
Substrate requirements for duplex DNA translocation by the eukaryal and archaeal minichromosome maintenance helicases
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DOI：
10.1074/jbc.m308599200
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发表时间：
2003-12-05
影响因子：
4.8
通讯作者：
Kelman, Z
中科院分区：
文献类型：
--
作者：
Shin, JH;Jiang, Y;Kelman, Z
复制型DNA解旋酶是环状的六聚体，其通过分离染色体DNA的两条链以提供复制型聚合酶的单链（ss）底物而在DNA合成中发挥重要作用。生物化学和结构研究表明，这些解旋酶沿着双链体的一条链沿着移位，该双链体穿过这些环形六聚体的中央通道并与之相互作用，并置换互补链。这些解旋酶中的许多也显示出同时环绕两条链，然后沿沿着双链（ds）DNA移位。本文报道了粟酒裂殖酵母Mcm 4，6，7复合体和嗜热甲烷杆菌MCM解旋酶沿双链DNA沿着移动。然而，这两种解旋酶在支持dsDNA易位所需的底物方面不同。虽然S. pombe Mcm 4，6，7复合物需要一个3 ′-突出端ssDNA区域来启动其与双链体的结合，古细菌蛋白在缺乏3 ′-突出端区域的情况下也启动其沿着dsDNA的转运。此外，含有链霉抗生物素蛋白生物素空间位阻的DNA底物抑制真核解旋酶的运动，沿着ss和dsDNA，但不是古细菌酶。分枝然而，热自养菌MCM解旋酶显示从ss以及dsDNA置换链霉抗生物素蛋白生物素复合物。在染色体复制的起始和延伸阶段的MCM蛋白的dsDNA易位的可能作用进行了讨论。
Replicative DNA helicases are ring-shaped hexamers that play an essential role in DNA synthesis by separating the two strands of chromosomal DNA to provide the single-stranded ( ss) substrate for replicative polymerases. Biochemical and structural studies suggest that these helicases translocate along one strand of the duplex, which passes through and interacts with the central channel of these ring-shaped hexamers, and displace the complementary strand. A number of these helicases were shown to also encircle both strands simultaneously and then translocate along double-stranded (ds) DNA. In this report it is shown that the Schizosaccharomyces pombe Mcm4,6,7 complex and archaeal minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus move along duplex DNA. These two helicases, however, differ in the substrate required to support dsDNA translocation. Although the S. pombe Mcm4,6,7 complex required a 3'-overhang ssDNA region to initiate its association with the duplex, the archaeal protein initiated its transit along dsDNA in the absence of a 3'-overhang region, as well. Furthermore, DNA substrates containing a streptavidinbiotin steric block inhibited the movement of the eukaryotic helicase along ss and dsDNAs but not of the archaeal enzyme. The M. thermautotrophicus MCM helicase, however, was shown to displace a streptavidinbiotin complex from ss, as well as dsDNAs. The possible roles of dsDNA translocation by the MCM proteins during the initiation and elongation phases of chromosomal replication are discussed.