Amaratunga M, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1993 Jul 13;32(27):6815-20. doi: 10.1021/bi00078a003.
DNA helicases unwind duplex DNA to form the single-stranded (ss) DNA intermediates required for replication, recombination, and repair in reactions that require nucleoside 5'-triphosphate hydrolysis. Helicases generally require a ss-DNA flanking the duplex in order to initiate unwinding in vitro; however, the precise function of the ss-DNA is not understood. If a helicase unwinds DNA by a "passive" mechanism, it would bind to and translocate unidirectionally along the ss-DNA and facilitate duplex unwinding by translocating onto the ss-DNA that is formed transiently by thermal fluctuations in the duplex. We have examined the kinetics of DNA unwinding by Escherichia coli Rep protein (a 3' to 5' helicase) by rapid quench-flow methods using a series of novel, nonnatural DNA substrates possessing 3' flanking ss-DNA within which is embedded either a segment of ss-DNA possessing reversed backbone polarity or a non-DNA [poly(ethylene glycol)] spacer, either of which should block unwinding by a passive helicase. The E. coli Rep helicase effectively unwinds these DNA substrates, ruling out a passive mechanism of unwinding. Instead, the results are consistent with an "active" rolling mechanism during which Rep binds to ss-DNA and duplex DNA simultaneously.
DNA解旋酶解开双链DNA，以形成复制、重组和修复所需的单链（ss）DNA中间体，这些反应需要核苷5'-三磷酸水解。解旋酶通常需要双链侧翼的单链DNA才能在体外启动解旋；然而，单链DNA的确切功能尚不清楚。如果解旋酶通过“被动”机制解开DNA，它将结合并沿单链DNA单向移位，并通过移位到由双链中的热波动瞬时形成的单链DNA上来促进双链解旋。我们使用一系列新型的非天然DNA底物，通过快速淬灭流动法研究了大肠杆菌Rep蛋白（一种3'至5'解旋酶）解开DNA的动力学，这些底物具有3'侧翼单链DNA，其中嵌入了一段具有反向主链极性的单链DNA片段或一个非DNA[聚（乙二醇）]间隔物，这两者都应能阻止被动解旋酶的解旋。大肠杆菌Rep解旋酶有效地解开了这些DNA底物，排除了被动解旋机制。相反，结果与“主动”滚动机制一致，在此过程中Rep同时结合单链DNA和双链DNA。
