Opening Closed Arms: Long-Distance Activation of SMC ATPase by Hinge-DNA Interactions  Michiko Hirano, Tatsuya Hirano  Molecular Cell  Volume 21, Issue 2, Pages 175-186 (January 2006) DOI: 10.1016/j.molcel.2005.11.026 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Molecular Architecture and the ATPase Cycle of SMC Proteins (A) Overall architecture of an SMC dimer. See text for details. (B) The SMC ATPase cycle. Binding of ATP (red) to the head domains induces their engagement, and hydrolysis of ATP triggers their disengagement. The Walker A mutation K37I prevents ATP binding, whereas the C motif mutation S1090R blocks engagement. The transition-state mutation E1118Q stabilizes engagement by slowing down ATP hydrolysis. The engaged heads can covalently be crosslinked to each other in constructs that contain two artificial cysteines, S55C and S1070C. (C) A dimerized SMC hinge based on the crystal structure of the TmSMC hinge (upper left). Shown are the positions of the four lysine residues that form a basic patch at the inner surface of the BsSMC hinge domain. Note that K565 comes from one subunit, (blue) whereas K666, K667, and K668 come from the other subunit (magenta). The right panel represents a view from the bottom, in which two basic patches can be seen. The positions of the four glycine residues critical for dimerization (G657, G658, G662, and G663) are shown in yellow. When these glycines are simultaneously mutated into glutamates, the resulting mutant protein DDDD fails to dimerize (lower panel), producing a single-armed monomer. (D) Sequence alignment of the hinge domain among different SMC proteins. Shown are: BsSMC, Bacillus subtilis SMC; MjSMC, Methanococcus jannaschii SMC; hSMC1-4, human SMC1-4. Conserved residues are shown in green, including the glycines critical for dimerization. Regions enriched in basic residues are shown in red. Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 A Basic Patch in the Hinge Domain Is Critical for Basal DNA Binding and DNA-Stimulated ATP Hydrolysis by SMC (A) A fixed concentration (15.6 μM nucleotides) of negatively supercoiled dsDNA (upper panels) or ssDNA (lower panels) was incubated with three different concentrations of proteins (105, 210, and 420 nM arms) in the presence or absence of ATP. No protein was added in lane 1. The reaction mixtures were fractionated on a 0.7% agarose gel and visualized by EtBr stain. Protein-DNA complexes are indicated by asterisks and free DNAs are indicated by arrows. (B) Purified proteins were loaded onto 5%–20% sucrose gradients and centrifuged at 189,000 × g (45,000 rpm) for 18 hr in an SW50.1 rotor. Fractions were subjected to SDS-PAGE and stained with Coomassie Blue. The positions of three protein standards (ovalbumin [3.7S], BSA [4.6S], and aldolase [7.3S]) are indicated. (C) ATPase activities of the four proteins were measured under different conditions. MgCl2 titration at 5 mM KCl (panels 1, 3, 5, and 7) and at 50 mM KCl (panels 2, 4, 6, and 8) in the presence of no DNA, ssDNA (31.2 μM nucleotides) or dsDNA (31.2 μM nucleotides). A fixed protein concentration of 300 nM arms was used for all the proteins. The rate of ATP hydrolysis is expressed as the number of ATP molecules hydrolyzed per second per arm. Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 ATP-Dependent DNA Manipulations Driven by Head-Head Engagement (A) Negatively supercoiled dsDNA was incubated with GGGG-E1118Q in buffers containing increasing concentrations of KCl (7.5, 10, 12.5, 25, 50, 75, and 100 mM) in the absence or presence of ATP. The reaction mixtures were analyzed as described in Figure 2A. (B) dsDNA was incubated with GGGG-E1118Q in a buffer containing 7.5 mM KCl in the absence or presence of ATP. After a 30 min incubation, the reaction mixtures were supplemented with increasing concentrations of apyrase (0.125–2.0 units/ml), incubated for another 30 min, and analyzed as above. (C) Linearized dsDNA was incubated with no protein (−), GGGG-K37I (WA), GGGG-S1090R (CM), or GGGG-E1118Q (TR) in the presence of ATP, and different concentrations of T4 DNA ligase were added as indicated. After a 30 min incubation, the DNA was isolated and fractionated on a 0.7% agarose gel. The positions of monomers, intramolecular (intra), and intermolecular (inter) end-end joining products are shown. (D) Negatively supercoiled dsDNA was incubated with the indicated set of proteins in the absence or presence of ATP, and then a restriction enzyme was added. After incubation, the DNA was isolated and analyzed as in (C). The positions of uncleaved, supercoiled DNA (SC) and cleaved liner DNA (L) are shown. (E) Relaxed circular DNA was incubated with the indicated set of proteins in the presence of ATP, and then calf thymus topoisomerase II was added. After incubation, the DNA was isolated and analyzed as in (C). (F) A fixed concentration of the wild-type protein GGGG (300 nM arms) was mixed with increasing concentrations (0–600 nM arms) of GGGG-K37I (panel 1), GGGG-S1090R (panel 2), or GGGG (panel 3). The ATPase activity of the mixtures was determined in the absence (no DNA) or presence of ssDNA or dsDNA. The final concentrations of KCl and MgCl2 were 15 mM and 2 mM, respectively. The activity is shown as a percentage of the “wild-type ratio = 1 (300 nM arms).” (G) dsDNA binding by GGGG-E1118Q and GGGG-E1118Q-K565E was assayed under the same conditions as described in Figure 2A. Protein-DNA complexes are indicated by a black asterisk, and free DNAs are indicated by an arrow. A small yet discrete shift supported by GGGG-E1118Q-K565E in the presence of ATP is indicated by a white asterisk (lane 13). (H) Ligation assays were performed in buffers containing increasing concentrations of KCl (7.5, 10, 12.5, 25, 50, 75, and 100 mM). Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Unusual End-End Joining Activities Associated with Open Hinge Mutants (A) The dsDNA and ssDNA binding activities of the open hinge mutants (AAAA, AAAA-K565E, and AAAA-K666E-K667E-K668E) were assayed under the same conditions as described in Figure 2A. (B) A ligation assay was performed with the indicated proteins under the same conditions as described in Figure 3H. Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 ATP-Driven Head-Head Engagement in Single-Armed Mutants (A) dsDNA binding by the indicated proteins was assayed under the same conditions as described in Figure 2A (upper panels). Note that DDDD-E1118Q and DDDD-E1118Q-K565E, but not DDDD, display a small yet discrete shift in an ATP- and dose-dependent manner (indicated by white asterisks in lanes 13 and 20). Alternatively, glutaraldehyde was added to a duplicated set of reactions at a final concentration of 0.2% and incubated for 10 min before fractionation (lower panels). Protein-DNA complexes are indicated by asterisks, and free DNAs are indicated by arrows. (B) Ligation assay was performed with the indicated proteins under the same conditions as described in Figure 3H. Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Requirement for Head-Head Disengagement in dsDNA Binding by SMC (A and B) In protocol number one, GGGG-E1118Q-2C or GGGG-E1118Q-0C was first treated without or with BMH in a buffer containing ATP for 30 min and then incubated with dsDNA for another 30 min. In protocol number two, the proteins were first incubated with dsDNA in the presence of ATP for 30 min and then treated without or with BMH for another 30 min. After quenching the crosslinking reaction, the reaction mixture was split into two aliquots. One was fractionated on an agarose gel and visualized by EtBr stain (A), and the other aliquot was analyzed by immunoblotting (B). Noncrosslinked polypeptides are indicated by an open triangle, and crosslinked products are indicated by an asterisk. (C) The same assay was performed as in (A) with DDDD-E1118Q-2C and DDDD-E1118Q-0C. Small yet discrete shifts observed are indicated by white asterisks (lanes 2, 4, 5, and 7–10). (D) Three different concentrations of GGGG-E1118Q-2C (105, 210, and 420 nM arms) were first treated without or with BMH in a buffer containing ATP for 30 min and then incubated with a fixed concentration of dsDNA (upper panels) or ssDNA (lower panels) for another 30 min. After quenching the crosslinking reaction, the reaction mixtures were analyzed as described above. Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Models for the Action of SMC Proteins (A) Wild-type BsSMC (GGGG) possesses a DNA-independent ATPase cycle that regulates engagement and disengagement of the two head domains within a dimer (stages 1′ and 3′). An initial interaction of SMC with DNA (the “sitting” mode) occurs at the hinge domain, guided by two coiled-coil arms (stages 1 and 2). This interaction triggers DNA-stimulated ATP hydrolysis and disengagement of the head domains (stage 3), thereby allowing the coiled-coil arms to fully open (stage 4). The basic-patch mutations K666E-K667E-K668E prevent the initial stage of SMC-DNA interaction regardless of the presence (from stage 1′ to 1) or absence (from stage 3′ to 3) of ATP. Opening of the hinge domain makes K565 accessible to dsDNA, leading to a stable DNA interaction by a hooking mechanism (stage 5). When ATP is available, it drives head-head engagement either intramolecularly by a trapping mode (stage 6) or intermolecularly by a gathering mode (stage 7). A variation of the latter mode could contribute to assembly of higher-order structures such as linear or helical filaments (stage 7′). The open-hinge mutant AAAA displays an unregulated dsDNA binding activity (stage 8). Although the single-armed mutant DDDD poorly binds to DNA (stage 9), it displays a unique DNA binding activity when ATP-driven head-head engagement is stabilized with the aid of E1118Q (stage 10). The reverse-V-shaped molecule may support DNA crosslinking by a mechanism similar to that proposed for Rad50 (stage 11). (B) A speculative view of how BsSMC might be loaded and unloaded during the bacterial chromosome cycle. See text for details. The yellow box indicates a postulated barrier of topological domains (Postow et al., 2001). Molecular Cell 2006 21, 175-186DOI: (10.1016/j.molcel.2005.11.026) Copyright © 2006 Elsevier Inc. Terms and Conditions