DNA binding ability of the Nse1-Nse3-Nse4 sub-complex of the SMC5/6 complex is essential in fission yeast

Smc5/6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. The Smc5/6 complex is essential for proliferation in yeast and is involved in the homologous recombination-based DNA repair processes, including repair of DNA double strand breaks, restart of stalled replication forks etc. However, the precise mechanism of SMC5/6 function is not known. We will present the evidence for direct physical interaction of its part, Nse1-Nse3-Nse4 sub-complex, to DNA and its essential role for the function of the whole SMC5/6 complex. The Nse1-Nse3-Nse4 sub-complex is rich in winged-helix domain motifs and at least two of them form a putative DNA-binding cleft. The purified Nse1-Nse3-Nse4 sub-complexes shift different DNA substrates in electromobility shift assays (EMSA) proving their ability to bind DNA in vitro. Mutations of the key basic residues within the putative DNA-binding cleft reduce in vitro binding to DNA. Introduction of these mutations into S. pombe genome results in cell death and/or hypersensitivity to hydroxyurea. The chromatin immunoprecipitaion (ChIP) analysis of the DNA-binding mutant shows reduced association of SMC5/6 with chromatin. The above data and our genetic analysis indicate the essential role of the interaction (between Nse1-Nse3-Nse4 and DNA) for the loading and/or maintenance of the SMC5/6 complex on the yeast chromatin.