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

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.

The SMC (Structural Maintenance of Chromosomes) complexes are key organizers of prokaryotic and eukaryotic genomes. They organize chromatin domains (cohesins 1 ), condense mitotic chromosomes (condensins 2 ), assist in DNA repair (SMC5/6 3,4 ) and replication (SMC/ScpAB 5 ). These circular complexes use the energy of ATP hydrolysis to drive DNA topology changes. In prokaryotes, SMC/ScpAB drives extrusion of loops behind the replication fork. In eukaryotes, condensins extrude loops laterally and axially to shape chromatin to the typical mitotic chromosomes. Cohesins assist in formation of topologically associating domains during interphase. Cohesin rings can also hold newly replicated sister chromatids together and release them in highly controlled manner. The SMC5/6 complexes have been implicated in the repair of DNA damage by homologous recombination, and in stabilization and restart of stressed replication forks. The SMC5/6 instability leads to the chromosome breakage syndrome in human 6 , however, the molecular mechanism of the SMC5/6 action is largely unclear.
All the SMC complexes are composed of three common categories of subunits: SMC, kleisin and kleisin-interacting proteins 7,8 . The SMC proteins are primarily built of long anti-parallel coiled-coil arms, a globular hinge (situated in the middle of the peptide chain) and a head domain (formed by combined amino and carboxyl termini [9][10][11][12][13] ). The globular head domain contains ATP binding and hydrolysis motifs of the ATP-binding cassette transporter family 14,15 . Two SMC molecules form dimers via the association of their hinge domains and transiently interact when their head domains sandwich a pair of ATP molecules. The binding of ATP changes conformation and shape of the SMC subunits at local as well as global levels 10 . At local level, the SMC heads and necks move from aligned position to the ATP-locked conformation. At global level, the overall shape of the complex changes from rod-to ring-like upon ATP binding (with heads locked by ATP at one end and hinge dimer at the other end). The hydrolysis of ATP dissolves the SMC-ATP-SMC head bridge.
The ATPase head domains are also connected by the kleisin subunit in an asymmetric way. Kleisin binds to the cap side of one SMC (designated as κSMC) head domain via a winged-helix domain (WHD) at its carboxyl terminus. Kleisin's α-helix located at its amino terminal helix-turn-helix (HTH) domain binds to the coiled-coil base region immediately adjacent to the other SMC head (called neck and designated as νSMC [16][17][18] ). This kleisin

Results
Nse4 interacts with SMC6 neck. We have previously shown that Nse4 belongs to the kleisin superfamily of proteins and binds to the SMC5 and SMC6 head fragments 28 . To map the Nse4 interaction with SMC6, we employed peptide library covering yeast S. pombe SMC6 region aa875-1024 (Supplementary Table 1). The peptides were pre-bound to ELISA plates and tested against Nse4(1-150) and control (human TRF2) proteins 29 . The peptides covering the C-terminal region of SMC6 (aa955-1009) bound to Nse4 (Suppl. Fig. S1A). The peptide aa960-984 exhibited the highest affinity and specificity to Nse4, while the other peptides bound to Nse4 in a less specific way (e.g. peptide aa970-994). Interestingly, the SMC6 region aa960-984 corresponds to the SMC neck regions interacting with kleisins in most SMC complexes 12 (Fig. 1A).
To distinguish mutations specifically disturbing the Nse4-SMC6 interaction from those affecting the multiple SMC6 interactions (e.g. by protein structure and stability alteration), we established another 3Y2H system consisting of the SMC6 and Nse5-Nse6 subunits (as Nse5 and Nse6 bind to the SMC6 protein 28 ). In this system, we used the same Gal4AD-SMC6 mutation constructs as above in combination with Gal4BD-Nse5 and p416ADH1-Nse6 (Figs. 1D and S1D). The L964A, L968A and E969A mutations reduced SMC6-Nse5-Nse6 complex stability, suggesting their deleterious effects on the SMC6 interactions in general (Fig. 1, compare panels B, C and D, columns 5, 7 and 8). Note that the protein levels of the SMC6/L968A and SMC6/E969A mutants were lower compared to the wild-type Gal4AD-SMC6, suggesting their destabilizing effect (Suppl. Fig. S1E, lanes 7 and 8). In contrast, the other mutations had no impact on the SMC6-Nse5-Nse6 stability, suggesting that the conserved L965, L972 and R975 residues within the SMC6 neck region mediate specifically the SMC6-Nse4 interaction (Figs. 1 and S1).
To analyse the role of the Nse4-SMC6 interaction in the fission yeast cells, we introduced the L62C and T65R mutations into the genome of diploid S. pombe. In diploid cells, the expression level of the FLAG-tagged nse4/ L62C, T65R was comparable to nse4/WT (Fig. 2E, lanes 2 and 3), however, the tetrad analysis showed that the double nse4-L62C, T65R mutation was lethal for haploid cells (Fig. 2E, middle panel), suggesting an essential role for the Nse4-SMC6 interaction.
A role for the Nse4 and ATP molecules in bridging of SMC5-SMC6. To compare the role of Nse4 and ATP in bridging of the SMC5-SMC6 heads, we introduced the SMC5/E995Q mutation which inhibits ATP hydrolysis; i.e. enhances ATP retention between SMC5-SMC6 heads and their dimerization (Figs. 3A and S3 14,35 ). In the SMC5-Nse4-SMC6 3Y2H system, the interaction between the Gal4BD-SMC5 and Gal4AD-SMC6 constructs was not detectable, suggesting a relatively low stability of the SMC5-SMC6 dimer even upon stable binding of ATP (Fig. 3A, columns 1 and 2). Addition of Nse4 resulted in relatively stable SMC5-Nse4-SMC6 complex formation (Fig. 3A, columns 3 and 4), suggesting that Nse4 stabilizes the bridge between SMC5 and SMC6. The introduction of the ATP-hydrolysis mutation to the SMC5-Nse4-SMC6 complex only slightly increased its relative stability (Fig. 3A, column 4), suggesting a major role of Nse4 (and a minor additive effect of the ATP binding; see below) in bridging of the SMC5-SMC6 heads.
When we reduced the Nse4 binding affinity to SMC6 using the specific Nse4 mutations described above, the stability of the wild-type SMC5 complexes dropped more dramatically than the stability of the SMC5/E995Q mutant complexes (Fig. 3A, compare odd and even columns). For example, the L68C mutation reduced stability of the wild-type SMC5-Nse4-SMC6 complex significantly, while the ATP molecule (in SMC5/E995Q) stabilized the Nse4/L68C complex (Fig. 3A, columns 5 and 6). Further reduction of the Nse4 binding affinity (Fig. 3A, columns 7-12) led to further drops in stability of SMC5-Nse4-SMC6, again, with relatively more stable hydrolytic mutants. Although the stability of the Nse4/L62C, T65R double mutant complex was very low, the residual affinity of Nse4 to SMC6 still supported ATP binding in the SMC5/E995Q mutant (Fig. 3A, columns 11 and 12). These data suggest that ATP contributes significantly to the SMC5-SMC6 bridging when the Nse4 affinity is reduced and that the Nse4 and ATP interactions are synergistic in the SMC5-Nse4-SMC6 complex.
Given that both ATP and Nse4 bridge SMC5-SMC6 heads, our data suggest that the ATP bridge antagonizes the KITE-bound Nse4 bridge (Fig. 3B, compare columns 3 and 4), and vice versa, the KITE-bound Nse4 bridge counteracts the ATP-mediated SMC5-SMC6 dimerization (Fig. 3B, compare columns 2 and 4). To release the constraint imposed by the KITE-bound Nse4 bridge, we reduced the Nse4 binding affinity to SMC6 using the specific Nse4 mutations described above (Figs. 2 and 3A). With reduction of the Nse4 affinity, the relative stability of the ATP-free complexes gradually dropped to its limit (Fig. 3A,B, columns 5, 7, 9 and 11) as the Nse4 bridge was the most dominant one. In contrast, the relative stability of the ATP-bound SMC5-SMC6-Nse4-Nse3-Nse1 complexes dropped first moderately in single mutants (Fig. 3B, columns 6, 8, and 10) and then it partially recovered in the L62C, T65R double mutant (column 12). The relative stability of the single nse4 mutants was less affected in the SMC5/E995Q background (compared to the SMC5/WT background) as the total ATP-bound complex stability was a result of the balance between the competing Nse4 and ATP bridges. In other words, with the weaker integrated into yeast S. pombe diploid cells and the following Nse4 protein levels were compared on western blot (left panel): Nse4/WT (lane2), nse4/ L62C, T65R (lane 3) and Nse4 with 30 amino acids extension of its linker region (lane 4). Tetrad dissection analysis of the nse4 + /nse4-L62C, T65R diploid strain shows (middle panel) that the nse4-L62C, T65R mutation is lethal, suggesting essential role of the Nse4-SMC6 interaction. The tetrad analysis of the nse4 + /nse4 ext diploid strain (right panel) suggests growth defect of the nse4/ext haploid cells. (2020) 10:9694 | https://doi.org/10.1038/s41598-020-66647-w www.nature.com/scientificreports www.nature.com/scientificreports/ Nse4 binding (and therefore lower complex stability), there was a weaker ATP-mediated constraint which allowed better ATP binding and improved complex stability. In the L62C, T65R double mutant, when the Nse4-SMC6 interaction was very weak, the ATP binding was only weakly opposed by the KITE-bound Nse4 bridge and ATP could bridge the SMC5-SMC6 heads efficiently (column 12).
Altogether, these data suggest that ATP constrains KITE-bound Nse4 bridge and vice versa, the KITE-bound Nse4 bridge counteracts the ATP-mediated SMC5-SMC6 heads dimerization, and that this constraint might be released via dissociation of the Nse4-SMC6 interaction (Fig. 3C).  1-4). The SMC5/E995 conserved residue was mutated to glutamine (EQ) to inhibit ATP hydrolysis. The ATP retention has only mild additive effect on the stability of the SMC5-Nse4-SMC6 complex (scored on plates containing increasing concentrations of 3-Amino-1,2,4triazole). The Nse4 mutations affect the stability of the wild-type SMC5 complexes more dramatically than the stability of the SMC5/E995Q mutant complexes (compare odd and even columns). Wild-type (WT) or E995Q (EQ) mutant versions of SMC5 are labelled in grey below the panels (further details as in Figs. 1 and 2). (B) Addition of the Nse1 and Nse3 KITE proteins to the above SMC5/SMC6/Nse4 system stabilizes the SMC5-Nse4-SMC6 bridge. Although the KITE proteins stabilize the ATP-free SMC5-Nse4-SMC6 complex (columns 1 and 3), they destabilize the ATP-bound complex (columns 2 and 4). The Nse4 mutations decrease stability of the ATP-free SMC5-SMC6-Nse4-Nse3-Nse1 complex gradually (columns 5, 7, 9 and 11), while the stability of the ATP-bound complexes drops first (columns 6, 8, and 10) and then it recovers in the L62C, T65R double mutant (column 12). (C) Schematic summary of the panel A and B results showing the stabilizing effect of the KITE binding to the Nse4 bridge and the destabilizing effect of ATP to the KITE-bound Nse4 bridge. The ATPimposed constraint is partially released via dissociation of the Nse4-SMC6 interaction.

Scientific RepoRtS |
(2020) 10:9694 | https://doi.org/10.1038/s41598-020-66647-w www.nature.com/scientificreports www.nature.com/scientificreports/ The ATP-mediated constraint depends on the KITE subunits. In the SMC5/6 complex, the KITE and kleisin subunits form a tight Nse1-Nse3-Nse4 sub-complex mediated by their mutual interactions (Fig. 4A) 28,34 . As ATP constrained the Nse4 bridge only in the presence of the KITE subunits (Fig. 3A,B, columns 3 and 4), we introduced mutations specifically affecting the stability of the Nse1-Nse3-Nse4 trimer to evaluate a role of the KITE proteins. Specific mutations disturbing only individual Nse1-Nse3 (Nse1/Q18A, M21A) and Nse3-Nse4 (Nse4/del87-91) binary interactions (Fig. 4A, compare columns 3 against 4 and 9 against 10) did not affect the stability of the whole Nse1-Nse3-Nse4 trimer (Fig. 4A, columns 6 and 7), but their combination compromised trimer assembly (Fig. 4A, column 8). When we introduced this combination of Nse1 and Nse4 mutations to the SMC5-SMC6-Nse4-Nse3-Nse1 complex, its stability was reduced as the KITE proteins lost their ability to bind The Nse1-Nse3-Nse4 subcomplex is held by mutual interactions between its subunits. The Nse4/del87-91 (del) deletion disturbs the Nse3-Nse4 binary interaction (columns 3 and 4) and the Nse1/Q18A, M21A (QM) mutation abrogates the Nse1-Nse3 binary interaction (columns 9 and 10), but they do not alter stability of the Nse1-Nse3-Nse4 trimer individually (columns 6 and 7). However, combination of these two mutations reduces the stability of Nse1-Nse3-Nse4 significantly (column 8). (B) The combination of the Nse4/del87-91 and Nse1/Q18A, M21A mutations compromises the stability of the wild-type SMC5-SMC6-Nse4-Nse3-Nse1 complex (compare columns 6 and 8), but increases the stability of the ATP-bound complex (compare columns 7 and 9). Notably, there is no difference between stability of the ATP-free and ATP-bound complexes (compare columns 8 and 9), suggesting that the binding of the KITE proteins to Nse4 destabilizes the ATP-bound complexes. Furthermore, the Nse4/del87-91 mutation alone also partially supresses the instability of the ATP-bound complex (column 11), suggesting that the binding of Nse3 to Nse4 linker constrains the linker. (C) Schematic summary of the results showing the constraint effect of ATP (Fig. 3) and its partial release via dissociation of the Nse3-Nse4 interaction. and stabilize Nse4 (Fig. 4B, compare columns 6 and 8). In contrast, when we introduced this combination of Nse1 and Nse4 mutations to the SMC5/E995Q hydrolytic mutant complex, the stability of the ATP-bound complex was increased (Fig. 4B, compare columns 7 and 9), suggesting that the ATP-induced constraint of Nse4 depends on its binding to the KITE subunits. Importantly, there was no difference between the stability of the ATP-free and ATP-bound complexes (compare columns 8 and 9), further corroborating our conclusion that the ATP-mediated constraint depends on the binding of the KITE dimer to Nse4. Furthermore, the Nse4/del87-91 mutation compromising only Nse3-Nse4 interaction (Fig. 4A, columns 4 and 6) had a suppressing effect on the SMC5/E995Q complex similar to the double mutant (Fig. 4B, compare columns 9 and 11), suggesting that the binding of Nse3 to the Nse4 linker partially constrained it. Our data suggest that the instability of the SMC5/6 core complex induced by the ATP binding is dependent on the binding of KITE proteins to the Nse4 kleisin linker and that the constraint can be partially released via dissociation of the Nse3-Nse4 interaction (Fig. 4C).
The limited size of the Nse4 linker imposes mechanical constraint. The KITE dimers bind the linker regions of kleisin molecules in the SMC complexes (Fig. 5A) 7,16,29,[38][39][40] . To explore the role of the Nse4 linker in propagation of the ATP-induced constraint, we inserted a 30 amino acid extension at the putative end of the linker (Figs. 5A and S4A) to lengthen the linker. The Nse4 extended construct bound the Nse1-Nse3 KITE proteins normally (Fig. 5A) and formed the SMC5-SMC6-Nse4-Nse3-Nse1 complex with the relative stability similar to that with the normal Nse4 construct (Fig. 5B, columns 3 and 4). Interestingly, combination of the Nse4 extended construct with the SMC5/E995Q ATP-hydrolysis mutant partially increased the relative stability of the SMC5-SMC6-Nse4-Nse3-Nse1 complex, suggesting that the extended Nse4 linker partially alleviated the ATP-induced constraint (Fig. 5B, columns 5 and 6).
To assess the importance of the Nse4 linker size and the effect of its extension, we also introduced the 30 amino acid extension into the genomic copy of the fission yeast Nse4. In yeast diploid cells, the expression level of the FLAG-tagged nse4/ext was comparable to nse4/WT (Fig. 2E, lanes 2 and 4), however, the tetrad analysis showed that the haploid cells were sick (Fig. 2E, right panel). In addition, the nse4/ext haploid cells exhibited severe hydroxyurea (HU) and methyl methane sulfonate (MMS) sensitivities similar to that of the smc6-74 hypomorphic mutant (Fig. 5C), suggesting an important role of the Nse4 linker size for the SMC5/6 function during DNA repair and replication.

Discussion
The kleisin subunits bridge the SMC heads in an asymmetric way and lock the SMC ring at its head side 12,13,16,18 . We have shown that Nse4 belongs to the kleisin superfamily of proteins and binds strongly to the κSMC5 head via its Nse4 C-terminal WHD 28 . However, we observed only weak binding of Nse4 to νSMC6 (Fig. S1A) 28 and other studies actually failed to show the Nse4-SMC6 interaction 31,32,41 . Here we developed several unique systems to prove and analyse the interaction between Nse4 and SMC6. We mapped the Nse4-SMC6 interface in detail and found that the Nse4-SMC6 interaction mode is similar to the other νSMC-kleisin interactions (Figs. 2 and S2C [16][17][18]25,27 ). Therefore, we assume that Nse4 bridges the SMC5-SMC6 proteins in a way similar to kleisins in the other SMC complexes, except that the Nse4 bridge is specifically modulated by the Nse1-Nse3 KITE subunits in the SMC5/6 complex (see below).
The kleisins lock the SMC rings that can embrace DNA or extrude a loop in an ATP-dependent way 42,43 . To release such entrapped DNA, the SMC-SMC or SMC-kleisin interface must be open. It was proposed that the νSMC-kleisin interface opens and serves as an exit gate for trapped DNA 19,44,45 . In the cohesin complex, the Scc1-SMC3 interface is opened upon ATP binding (in the presence of the Pds5 and Wapl regulators [19][20][21][22][23][24]. Our data show that the SMC5/6 core complex is relatively less stable in the ATP-bound state than in the ATP-free state, suggesting that one or more protein-protein interaction interfaces are compromised upon ATP binding (Fig. 5D). Given the weak nature of the Nse4-SMC6 interaction, we assume that this interaction is prone to dissociation and that the Nse4-SMC6 interface is opened upon ATP binding. Consistent with the latter notion, the ATP-mediated constraint was released when the Nse4-SMC6 interaction was disturbed (Fig. 3B). Therefore, we hypothesize that the binding of ATP to the SMC5-SMC6 heads constrains the Nse4 bridge (when bound by the Nse1 and Nse3 KITE subunits; see below and Fig. 5D) and this constraint is partially released via dissociation of the Nse4-SMC6 interaction.
The ATP binding induces changes in the mutual positions (and conformations) of the SMC heads and arms 10,27,[46][47][48] . Consequently, the shape of the whole SMC complex changes from a rod-like conformation (with juxtaposed arms stabilized by their mutual interactions) to the less stable open ring (Fig. 5D). As for the other SMC complexes, we hypothesize that at least part of the ATP-induced SMC5/6 instability (Fig. 5D) might be a consequence of such SMC-SMC shape transition from the rod to ring. However, we observed this instability only in the presence of the KITE subunits, suggesting that either our system is unable to detect the SMC5-SMC6 rod-to-ring shape transition (and monitors only Nse4 bridge opening) or the KITE proteins are required for the full rod-to-ring shape transition. The latter possibility is consistent with the prokaryotic SMC/ScpAB data which suggest an important function of the KITE subunits in pulling SMC arms 26,27 ; however, further experiments with purified SMC5/6 complexes are needed to resolve this issue. In our hypothetical model, the binding of ATP to the SMC5-SMC6 heads pulls their arms apart and constrains the KITE-bound Nse4 bridge (Fig. 5D).
It was shown that kleisin-interacting proteins (particularly KITE and HAWK subunits) bind and shape linker regions of the kleisin molecules 7,11,16,29,[38][39][40][49][50][51] . Our data showed that the binding of the Nse1-Nse3 KITE dimer increases the Nse4 ability to bind SMC5-SMC6 subunits in ATP-free state (Figs. 3B and 4B), suggesting that the KITE binding may stabilize and shape Nse4 to fit the ATP-free conformation of the SMC5/6 core complex. In contrast, the KITE-bound Nse4 linker is less compatible with the ATP-bound conformation of SMC5/6. Consistent with these notions, extensions of the Nse4 linker partially relaxed its stiff shape and resulted in the reduced stability of the ATP-free complexes (Figs. 5B and S4). On the contrary, the linker extensions partially released the Scientific RepoRtS | (2020) 10:9694 | https://doi.org/10.1038/s41598-020-66647-w www.nature.com/scientificreports www.nature.com/scientificreports/ The schematic summary of the roles of the KITE subunits in the ATPase cycle of SMC5/6. Our data show that the SMC5/6 core complex is less stable in the ATP-bound state than in the ATP-free state. We hypothesize that the KITE-shaped Nse4 linker fits the ATP-free conformation of SMC5/6 (and therefore increases its stability), while the ATP-bound conformation is less compatible with the KITE-shaped Nse4 linker (and therefore constrains the Nse4 bridge). The reduced stability of the ATP-bound complex may suggest that one or more protein-protein interaction interfaces are compromised upon ATP binding. The results in the Fig. 3 suggest that the Nse4-SMC6 interface might be open. The results in the Fig. 4 also point to the Nse4-Nse3 interface. In addition, the interface between SMC5-SMC6 arms might be disturbed upon the ATP binding as suggested for the other SMC complexes. The stiff KITE-bound Nse4 linker may transduce a pulling force generated by the binding of ATP to the SMC5-SMC6 heads. In consequence, the Nse4-SMC6 and Nse4-Nse3 interfaces may open and release the Nse4 constraint. (2020) 10:9694 | https://doi.org/10.1038/s41598-020-66647-w www.nature.com/scientificreports www.nature.com/scientificreports/ ATP-induced constraint in the ATP-bound complexes. Therefore, we suggest that the KITE subunits shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding (Fig. 5D). Consistent with this conclusion, the ATP-mediated constraint was partially suppressed upon release of the part of the Nse4 linker from the Nse3 binding pocket (Fig. 4B) 29,34 . Altogether, we hypothesize that the Nse4 linker is stiffened upon its KITE binding and transduces a pulling force generated by the binding of ATP to the SMC5-SMC6 heads (Fig. 5D). In consequence, the Nse4-SMC6 interface opens and releases the Nse4 constraint.
Similarly, it was proposed that binding of the HAWK (Scc3 and Pds5) and Wapl proteins to the Scc1 kleisin stiffens its linker region and transduces conformational energy of the ATP-dependent SMC head dimerization to the dissociation of Scc1 from Smc3 19,45 . In the absence of the Pds5-Wapl regulators, the cohesin's head movements driven by the ATP binding and hydrolysis cannot be effectively coupled to exit gate opening as the Scc1 linker is flexible. Interestingly, the size of the Scc1 linker is much longer (cca 400 amino acids) than the size of the Nse4 linker). Accordingly, the Scc3 HAWK subunit covers only a small part of the Scc1 linker and requires Pds5-Wapl regulators to shape the long Scc1 linker, while the KITE subunits are sufficient to shape their short kleisin partners 27,38 . As mentioned above, the extension of the Nse4 linker region suppressed the ATP-induced constraint, suggesting that the short size of the Nse4 linker is critical for the dynamics of the SMC5/6 complex (Figs. 5 and S4D). Consistent with this notion, the integration of the 30 amino acid long extension to the genomic copy of the fission yeast Nse4 resulted in the severe DNA repair phenotypes (Fig. 5C).
Taken together, we propose a hypothetical model in which the KITE proteins shape the kleisin linker connecting the SMC heads (Fig. 5D). The KITE-shaped Nse4 linker fits the ATP-free conformation of SMC5/6 (and therefore increases its stability), while the ATP-bound conformation is less compatible with the KITE-shaped Nse4 linker (and therefore constrains the Nse4 bridge). This hypothetical model suggests a key role of the kleisin and KITE subunits in the molecular mechanisms driving the SMC5/6 dynamics.
To prepare p416ADH1-Nse4/ext, SalI restriction site in the p416ADH1 multi-cloning site (MCS) was mutated by site-directed mutagenesis (SDM; see below) with oLV522 + oLV523 (Suppl. Table 3). Then, SalI site was inserted by SDM behind the aa174 with oLV520 + oLV521. This construct was SalI digested, (G 4 S) 6 linker was amplified by oLV579 + oLV580 and inserted using the In-Fusion cloning protocol.
Construct for the S. pombe genome integration was prepared as follows: 1. Nse4 was cloned within the BamHI and EcoRI sites of the pSK-ura4 plasmid; 2. genomic sequence downstream of the Nse4 gene was PCR amplified (JP414 and JP415) and inserted to the pGEM-Easy vector (Promega, USA); 3. The SacI-SalI fragment of the pSK-Nse4-ura4 plasmid was inserted to the SacI-XhoI digested pGEM-3′end construct to get the pGEM-Nse4(WT)-ura4 integration plasmid. To create pGEM-Nse4(L62C, T65R)-ura4 and pGEM-Nse4/ext, the Nse4 sequences were PCR amplified from the p416ADH1-Nse4 constructs by oLV680 + oLV681 (Supplementary Table 2) and inserted into BamHI-EcoRI digested pGEM-Nse4(WT)-ura4 integration construct by the In-Fusion cloning protocol. Additionally, the NdeI site was introduced next to the Nse4 ORF (using oLV689 + oLV690 primers for SDM) and three copies of FLAG-tag (amplified by oLV691 + oLV692 primers) were inserted to the NdeI site.