Chan Ying Wai, West Stephen C
The Francis Crick Institute, London, United Kingdom.
The Francis Crick Institute, London, United Kingdom.
Methods Enzymol. 2018;600:527-542. doi: 10.1016/bs.mie.2017.11.020. Epub 2018 Jan 9.
Successful chromosome segregation depends on the timely removal of DNA recombination and replication intermediates that interlink sister chromatids. These intermediates are acted upon by structure-selective endonucleases that promote incisions close to the junction point. GEN1, a member of the Rad2/XPG endonuclease family, was identified on the basis of its ability to cleave Holliday junction recombination intermediates. Resolution occurs by a nick and counter-nick mechanism in strands that are symmetrically related across the junction point, leading to the formation of ligatable nicked duplex products. The actions of GEN1 are, however, not restricted to HJs, as 5'-flaps and replication fork structures also serve as excellent in vitro substrates for the nuclease. In the cellular context, GEN1 activity is observed late in the cell cycle, as most of the protein is excluded from the nucleus, such that it gains access to DNA intermediates after the breakdown of nuclear envelope. Nuclear exclusion ensures the protection of replication forks and other DNA secondary structures important for normal metabolic processes. In this chapter, we describe the purification of recombinant GEN1 and detail biochemical assays involving the use of synthetic DNA substrates and cruciform-containing plasmids.
成功的染色体分离依赖于及时清除连接姐妹染色单体的DNA重组和复制中间体。这些中间体由结构选择性核酸内切酶作用，促进在连接点附近进行切割。GEN1是Rad2/XPG核酸内切酶家族的成员，它是根据其切割霍利迪连接体重组中间体的能力而被鉴定出来的。切割通过在连接点两侧对称相关的链上的切口和反切口机制发生，导致形成可连接的带切口双链产物。然而，GEN1的作用并不局限于霍利迪连接体，因为5'-翼片和复制叉结构也是该核酸酶出色的体外底物。在细胞环境中，GEN1活性在细胞周期后期被观察到，因为大部分蛋白质被排除在细胞核外，使其在核膜破裂后能够接触到DNA中间体。核排除确保了对复制叉和其他对正常代谢过程重要的DNA二级结构的保护。在本章中，我们描述了重组GEN1的纯化，并详细介绍了涉及使用合成DNA底物和含十字形结构质粒的生化分析。
