The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.

1 0 1 1 Keywords: SMC5/6 complex; Nse4 klesin linker; Nse1/Nse3 KITE subunits; ATP binding; 1 2 protein-protein interaction, fission yeast 1 3 1 4 2 ABSTRACT 1 5 The SMC (Structural Maintenance of Chromosomes) complexes are composed of 1 6 SMC dimers, kleisin and kleisin-interacting subunits. Mutual interactions of these subunits 1 7 constitute the basal architecture of the SMC complexes. Particularly, terminal domains of the 1 8 kleisin subunit bridge the SMC head domains of the SMC molecules. Binding of ATP 1 9molecules to the heads and their hydrolysis alter the shape of long SMC molecules (from rod-2 0 3 1 upon ATP binding. This mechanism suggests an important role of the KITE subunits in the 3 2 dynamics of the SMC5/6 complexes. 3 3 3 4 AUTHOR SUMMARY 3 5The SMC5/6 complex is member of the Structural maintenance of chromosomes 3 6 (SMC) family, key organizers of both prokaryotic and eukaryotic genomes. Their architecture 3 7 and dynamics (driven by ATP binding and hydrolysis) are essential for cellular processes, like 3 8 3 chromatin segregation, condensation, replication and repair. In this paper, we described 3 9 conserved mode of the Nse4 kleisin subunit binding to the SMC6 (similar to cohesin and 4 0 condensin) and its bridging role. Furthermore, we showed different impact of the binding of 4 1 the Nse1-Nse3 KITE subunits to the Nse4 kleisin bridge. Our study suggested that the KITE 4 2 proteins modulate the stability of the SMC5/6 complex, depending on its binding and 4 3 hydrolysis of ATP. Our findings uncover molecular mechanisms underlying dynamics of the 4 4 SMC5/6 complexes.4 5 4 6 INTRODUCTION 4 7 The SMC (Structural Maintenance of Chromosomes) complexes are key organizers of 4 8 prokaryotic and eukaryotic genomes. They organize chromatin domains (cohesins; [1]), 4 9 condense mitotic chromosomes (condensins; [2]), assist in DNA repair (SMC5/6; [3, 4]) and 5 0 replication (SMC/ScpAB; [5]). These circular complexes use the energy of ATP hydrolysis to 5 1 drive DNA topology changes. In prokaryotes, SMC/ScpAB drives extrusion of loops forming 5 2 behind the replication fork. In eukaryotes, condensins extrude loops laterally and axially to 5 3 shape chromatin to the typical mitotic chromosomes. Cohesins assist in formation of 5 4 topologically associating domains during interphase. Cohesin rings can also hold newly 5 5 replicated sister chromatids together and release them in highly controlled manner. The 5 6 SMC5/6 complexes have been implicated in the repair of DNA damage by homologous 5 7 recombination, stabilization and restart of stressed replication forks. The SMC5/6 instability 5 8 leads to the chromosome breakage syndrome in human [6], however, the molecular 5 9mechanism of the SMC5/6 action is largely unclear. 6 0 All the SMC complexes are composed of three common categories of subunits: SMC, 6 1 kleisin and kleisin-interacting proteins [7, 8]. The SMC proteins are primarily build of long 6 2 anti-parallel coiled-coil arms, a globular hinge (situated in the middle of their peptide chain) 6 3 4...