Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase
Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase
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DOI：
10.1038/nsmb1055
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发表时间：
2006-03-01
影响因子：
16.8
通讯作者：
Raney, KD
中科院分区：
文献类型：
--
作者：
Eoff, RL;Raney, KD
解旋酶在 ATP 依赖性反应中的复制、修复和重组过程中解旋双链 DNA。可以使用寡核苷酸底物测量从双链 (ds) DNA 形成单链 (ss) DNA 的过程来研究解旋酶活性的机制。该测定提供了“全有或全无”读数，因为未检测到部分解开的中间体。我们已经确定了可以通过捕获部分解开的底物来检测 Dda 解旋酶反应循环中的中间体的条件。该中间体的出现支持了一种模型，其中每个 ssDNA 产物在解旋发生后与解旋酶相互作用。动力学分析表明，中间体出现在反应循环的缓慢步骤中，其两侧是更快的解旋步骤。这些观察结果证明了单体解旋酶包含不均匀步骤的复杂机制。讨论了这种机制的潜在生物学意义。
Helicases unwind dsDNA during replication, repair and recombination in an ATP-dependent reaction. The mechanism for helicase activity can be studied using oligonucleotide substrates to measure formation of single-stranded ( ss) DNA from double-stranded ( ds) DNA. This assay provides an 'all-or-nothing' readout because partially unwound intermediates are not detected. We have determined conditions under which an intermediate in the reaction cycle of Dda helicase can be detected by trapping a partially unwound substrate. The appearance of this intermediate supports a model in which each ssDNA product interacts with the helicase after unwinding has occurred. Kinetic analysis indicates that the intermediate appears during a slow step in the reaction cycle that is flanked by faster steps for unwinding. These observations demonstrate a complex mechanism containing nonuniform steps for a monomeric helicase. The potential biological significance of such a mechanism is discussed.