Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
Methods. 2022 Aug;204:348-360. doi: 10.1016/j.ymeth.2021.12.002. Epub 2021 Dec 8.
There are multiple assays available that can provide insight into the biochemical mechanism of DNA helicases. For the first 22 years since their discovery, bulk-phase assays were used. These include gel-based, spectrophotometric, and spectrofluorometric assays that revealed many facets of these enzymes. From 2001, single-molecule studies have contributed additional insight into these DNA nanomachines to reveal details on energy coupling, step size, processivity as well as unique aspects of individual enzyme behavior that were masked in the averaging inherent in ensemble studies. In this review, important aspects of the study of helicases are discussed including beginning with active, nuclease-free enzyme, followed by several bulk-phase approaches that have been developed and still find widespread use today. Finally, two single-molecule approaches are discussed, and the resulting findings are related to the results obtained in bulk-phase studies.
有多种分析方法可用于深入了解 DNA 解旋酶的生化机制。在发现后的 22 年中，一直使用的是批量分析方法。这些方法包括基于凝胶的、分光光度法和荧光分光光度法，这些方法揭示了这些酶的许多方面。从 2001 年开始，单分子研究为这些 DNA 纳米机器提供了更多的见解，揭示了能量偶联、步幅、进程以及单个酶活性的独特方面的细节，这些细节在集合研究中固有的平均化中被掩盖了。在这篇综述中，讨论了研究解旋酶的重要方面，包括从活性、无核酸酶的酶开始，然后讨论了几种已开发并仍在广泛使用的批量分析方法。最后，讨论了两种单分子方法，并将得到的结果与批量研究的结果联系起来。
