A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6

Abstract

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...

DNA segment capture by Smc5/6 holo-complexes

“…The neck gate interface also appears labile in purified preparations of cohesin and condensin (sub-)complexes with the binding partners detaching from one another upon ATP-head engagement 65,67 [or incubation with cohesin unloading factors 68 ]. A similar reaction has been proposed for fission yeast Smc5/6 based on yeast-twohybrid experiments 69 . Neck gate opening upon ATP binding and head engagement, however, diametrically contrasts with our finding on budding yeast Smc5/6, where ATP-dependent head engagement leads to closure rather than opening of the gate ( Fig 7 ).…”

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

“…Multiple inter-domain cross-links of the Nse1/3/4 module to the Smc5 and Smc6 proteins, mostly to the heads, were lost upon ATP and plasmid DNA addition. We postulate that Nse1/3/4 relocates by binding to DNA using the proposed DNA-binding residues in Nse3 (Zabrady et al, 2016;Vondrova et al, 2020), possibly assisting to evict Nse5/6 from the heads and if so underscoring the central role of Nse5/6 in regulating the enzymatic activities of the Smc5/6 complex.…”

“…Another invariably conserved subunit, called “kleisin”, asymmetrically bridges the two SMC proteins at their head domains to create a tripartite ring structure capable of entrapping DNA in its lumen (Haering et al , 2004; Palecek et al , 2006; Burmann et al , 2013; Gligoris et al , 2014; Wilhelm et al , 2015). Kleisin also serves as an attachment point for additional proteins from the KITE (Kleisin‐Interacting Tandem winged‐helix Element) or HAWK (HEAT‐protein Associated With Kleisin) families (Palecek & Gruber, 2015; Wells et al , 2017) for which functions related to DNA substrate interactions and ATPase regulation are emerging (Zabrady et al , 2016; Kschonsak et al , 2017; Li et al , 2018; Vondrova et al , 2020).…”

Smc5/6, an atypical SMC complex with two RING-type subunits

“…Smc5/6 can also topologically associate with DNA, using the same ATP-dependent mechanisms as other SMC complexes [45]. This association probably involves switching between the recently observed open and closed arm conformations of the complex and may require the ATP-dependent aperture of the Nse4-Smc6 interface [27,38].…”

“…On the other hand, the conformational changes depend on the ATPase cycle: binding of ATP engages the ATPase heads and the SMC arms adopt an open conformation [25]; upon hydrolysis of ATP, the heads change their conformation, closing the arm space between the two SMC subunits [23,25] (Figure 1D). To allow the topological association of the complex with DNA, the ATPase activity needs to be exquisitely coordinated with opening of a protein-protein gate in the SMC complex, most probably at the kleisin-SMC interface [17,26,27]. Finally, the whole process must be very dynamic, allowing SMC complexes to alternate between different DNA binding interfaces and gradually expand loops.…”