Structure Basis for Shaping the Nse4 protein by the Nse1 and Nse3 dimer within the Smc5/6 complex

Abstract

The Structural Maintenance of Chromosome (SMC) family is a highly conserved group of protein complexes maintaining genomic stability in all kingdoms of life. Smc5/6 complex, one of eukaryotic SMC family complex, is primarily involved in the resolving topological constraints during replication fork progression and repairing double strand breaks via homologous recombination. Within this complex, a subcomplex composed of Nse1, Nse3 (KITE) and Nse4 (kleisin) is thought to play multiple roles through DNA binding and regulating ATP-dependent interaction between Nse4 and Smc5/6 to open DNA entry gate. Mutations in Nse3 are related with rare immune lung disease immunodeficiency and chromosome breakage syndrome (LICS). However, how the Nse1-Nse3-Nse4 subcomplex functions in these multiple capacities remain unclear. To address this question, I determined the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 complex at 1.7 Å resolution and examined how it interacts with DNA in vitro. The structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation, forming three extensive interfaces with Nse4 and forcing Nse4 into a Z-shaped conformation. Thereby, the compacted Nse4 conformation can disrupt the Nse4-Smc6 interface allowing DNA entry to the Smc5/6 complex. The Nse1-Nse3-Nse4 complex structure provides a molecular explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations in Nse3 could dislodge Nse4 from Nse1-Nse3. The DNA binding and mutagenesis analyses further show that in addition to Nse3, the N-terminal and the middle region of Nse4 are also involved in DNA binding and the mutations of those DNA binding residues in Nse4 effect cell viability. Integrating these data with previous crosslink mass spectrometry data and the DNA-bound cohesin structure, I propose a model of Smc5/6 complex and potential roles of the Nse1-Nse3-Nse4 complex in DNA binding and guiding DNA entry into the Smc5/6 complex.