Shah Punatar Rajvee, West Stephen C
The Francis Crick Institute, London, United Kingdom.
The Francis Crick Institute, London, United Kingdom.
Methods Enzymol. 2018;600:569-590. doi: 10.1016/bs.mie.2017.11.022. Epub 2018 Jan 9.
Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1.
霍利迪连接体在重组DNA分子之间提供共价连接，并且在有丝分裂时染色体分离之前需要被移除。其解离缺陷会导致有丝分裂灾难，其特征是形成DNA断裂和染色体畸变。从噬菌体到细菌、酵母和人类，在所有生命形式中都已鉴定出可解离重组中间体的酶。在高等真核生物中，霍利迪连接体由GEN1（一种在机制上类似于原型解离酶大肠杆菌RuvC的核酸酶）和SMX三核酸酶复合体解离。通过使用由RecA介导的链交换产生的质粒大小的DNA重组中间体，对这些酶的研究得到了促进。在这里，我们详细介绍这些类似于α结构的重组中间体的制备及其被RuvC和GEN1解离的过程。
