DNA Recombination and Repair Laboratory, Francis Crick Institute, London NW1 1AT, United Kingdom.
Proc Natl Acad Sci U S A. 2022 May 3;119(18):e2123420119. doi: 10.1073/pnas.2123420119. Epub 2022 Apr 22.
Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.
四链 DNA 中间体，也称为 Holliday 连接点（HJs），在同源重组和 DNA 修复过程中形成，其解旋对于正确的染色体分离是必要的。为了促进 HJ 加工的生化分析，我们开发了一种涉及 DNA 酶自我切割的方法，生成含有单 Holliday 连接点（sHJ）或双 Holliday 连接点（dHJ）的 1.8kb DNA 分子。我们表明，dHJ DNA（称为 HoJo DNA）被人 BLM-TopIIIα-RMI1-RMI2 复合物溶解，形成两个非交叉产物。然而，结构选择性内切酶（人 GEN1 和 SMX 复合物）将含有单或双 HJ 的 DNA 解析为产生交叉和非交叉产物的混合物。最后，我们证明染色质抑制 GEN 或 SMX 对双 HJ 的解析，而允许 BTRR 介导的溶解。
