A protocol to determine the activities of human MUS81-EME1&2 endonucleases.
A protocol to determine the activities of human MUS81-EME1&2 endonucleases.
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DOI：
10.1016/j.xpro.2022.101528
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发表时间：
2022-09-16
期刊：
影响因子：
--
通讯作者：
Lin, Zhonghui
中科院分区：
文献类型：
--
作者：
Fang, Qianqian;Hua, Zhengkang;Lin, Zhonghui
人MUS 81-EME1&2复合物是结构选择性核酸内切酶，在DNA损伤修复中发挥重要作用。在这里，我们描述了一个协议，以确定对各种DNA结构的MUS 81-EME1&2复合物的核酸内切酶活性。我们共表达了MUS 81与EME 1或EME 2，并纯化了高纯度的复合物，并测定了它们对3个折叠瓣、5个折叠瓣、切口双链DNA和Holliday接头的切割活性。该方案也可用于确定其他结构选择性核酸内切酶的底物偏好。有关本方案使用和执行的完整详细信息，请参阅。人MUS 81-EME1&2复合物的表达和纯化MUS 81-EME1&2复合物的核酸内切酶活性的基于凝胶的测量MUS 81-EME1&2核酸内切酶的底物偏好的定量评估出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。人MUS 81-EME1&2复合物是结构选择性核酸内切酶，在DNA损伤修复中发挥重要作用。在这里，我们描述了一个协议，以确定对各种DNA结构的MUS 81-EME1&2复合物的核酸内切酶活性。我们共表达了MUS 81与EME 1或EME 2，并纯化了高纯度的复合物，并测定了它们对3个折叠瓣、5个折叠瓣、切口双链DNA和Holliday接头的切割活性。该方案也可用于确定其他结构选择性核酸内切酶的底物偏好。
The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their activities on the cleavages of 3ˊ flaps, 5ˊ flaps, nicked double-stranded DNAs, and Holliday junctions. This protocol can also be used for the determination of substrate preferences of other structure-selective endonucleases. For complete details on the use and execution of this protocol, please refer to. Expression and purification of human MUS81-EME1&2 complexes Gel-based measurement of the endonuclease activities of MUS81-EME1&2 complexes Quantitative evaluation of the substrate preference of MUS81-EME1&2 endonucleases Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their activities on the cleavages of 3ˊ flaps, 5ˊ flaps, nicked double-stranded DNAs, and Holliday junctions. This protocol can also be used for the determination of substrate preferences of other structure-selective endonucleases.
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