Eoff Robert L, Raney Kevin D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, Arkansas 72205, USA.
Nat Struct Mol Biol. 2006 Mar;13(3):242-9. doi: 10.1038/nsmb1055. Epub 2006 Feb 12.
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.
解旋酶在复制、修复和重组过程中通过依赖ATP的反应解开双链DNA（dsDNA）。可以使用寡核苷酸底物来研究解旋酶活性的机制，以测量双链DNA（dsDNA）形成单链（ss）DNA的情况。该检测提供了一种“全或无”的读数，因为未检测到部分解旋的中间体。我们已经确定了在何种条件下，可以通过捕获部分解旋的底物来检测Dda解旋酶反应循环中的中间体。这种中间体的出现支持了一种模型，即每个单链DNA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一个缓慢步骤中，该步骤两侧是更快的解旋步骤。这些观察结果证明了单体解旋酶的机制包含不均匀步骤。讨论了这种机制潜在的生物学意义。
