Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
Nucleic Acids Res. 2019 Feb 28;47(4):1935-1949. doi: 10.1093/nar/gky1280.
Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.
人类 GEN1 是一种胞质同源重组蛋白，在核膜溶解后能解决持续存在的四向 Holliday 连接（HJ）。已经有人提出 GEN1 二聚化在 HJ 的解决中起着关键作用，但它的反应动力学细节仍然难以捉摸。在这里，单分子 FRET 显示了人类 GEN1 如何结合 HJ，并总能确保在 GEN1-HJ 复合物的寿命内解决它。GEN1 单体通常在其初始结合中遵循 HJ 的异构化偏好，随后为催化而扭曲它。GEN1 单体保持紧密结合，没有明显的解离，直到 GEN1 二聚体形成并且 HJ 完全解决。GEN1 二聚体的快速 ON 和缓慢 OFF 速率及其对单链切割 HJ 的增加亲和力强制向前反应。此外，如果 GEN1 二聚体或其单体之一在第一次切割后解离，GEN1 单体对单链切割 HJ 的紧密结合提供了一种故障安全机制。GEN1 单体对完整和单链切割 HJ 的紧密结合使它成为处理逃避主要机制的 HJ 的最后手段。
