A model for Scc2p stimulation of cohesin's ATPase and its inhibition by acetylation of Smc3p

Abstract

The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p-Smc3p interface.

Keywords: ATPase; ECO1; ESCO1; NIPBL; SCC2; SMC; acetylation; cohesin; cohesion.

Figure 1.

Characterization of the acetyl mimic and its suppression by Scc2p/Scc4p overexpression. (A) Cartoon depiction of cohesin (Smc1p, Smc3p, Scc3p, and Mcd1p) with the associated stimulatory factor Scc2p (NIPBL). (B) Cryo-EM structure of S. cerevisiae cohesin (PDB ID: 6ZZ6) (Collier et al. 2020) illustrating that Smc3p-K113 acetylation eliminates a salt bridge with Scc2p-E822. (Magenta) Smc3p-K113, (salmon) Scc2p-E822, (yellow) distance for a putative salt bridge. The top panels show that a salt bridge can form between Smc3p-K113 (left) or the K113R mutation (right) and Scc2p-E822 due to close proximity. The bottom panels illustrate that Smc3p-K113 acetylation (left) or K113Q mutation (right) alters spacing, precluding a salt bridge. All structure illustrations in this report, including residue substitutions and alterations, were generated using PyMOL software. (C) SCC2/SCC4 overexpression suppresses the inviability of the smc3-K113Q acetyl mimic. The haploid SMC3-AID strain also bearing either a wild-type SMC3 (VG3919-3C), smc3-K113Q (KB62A), or smc3-K113Q containing pGAL-SCC2/SCC4 (VG4052-3A) was grown at 30°C to saturation; plated at 10-fold serial dilutions on YPD, YPD + IAA, or YEPG + IAA; and incubated for 4 d at 23°C. (D) SCC2/SCC4 overexpression fails to suppress the cohesion defect of smc3-K113Q cells. Strains in C, along with a haploid containing SMC3-AID as the sole SMC3 (VG3651-3D), were arrested in G1, depleted for SMC3-AID, SCC2/SCC4-overexpressed, synchronously released from G1, and arrested in mid-M phase. Cells were fixed and processed to score cohesion at a chromosome IV arm locus using the LacO–LacI system as described in the Materials and Methods. The number of GFP spots was scored in mid-M-phase-arrested cells, and the percentage of cells with defective cohesion (two GFP spots) was plotted. Two-hundred cells were scored for each data point, and data were generated from two independent experiments. (E) Cohesin binding to chromosomes is greatly reduced in smc3-K113Q cells and only slightly increased by SCC2/SCC4 overexpression. Aliquots of mid-M-phase cells from D were fixed and processed for ChIP using anti-Mcd1p antibodies as described in the Materials and Methods. Mcd1p binding was assessed by qPCR and is presented as a percentage of input DNA. (Left panel) The chromosome IV arm CAR region (TRM1). (Middle panel) The chromosome III pericentric region (CARC1). (Right panel) Regions immediately flanking CEN14 and CEN4.

Figure 2.

Suppressor screen linking the Scc2p–Smc3p-K113 interface with other Scc2p–cohesin head interfaces. Identification of suppressor mutations in SMC1 and SMC3 that suppress the lethality of smc3-K113Q. (A) Schematic of the genetic screen used to identify smc3-K113Q suppressors. The haploid strain (VG3969-14C) bears smc3-K113Q as the sole S. cerevisiae copy of SMC3 and S. bayanus SMC3 on a CEN URA3 G418 plasmid (pFC3). Single colonies were grown to saturation in YPD and plated on FOA media to select for viable cells expressing only Smc3p-K113Q (see the Materials and Methods). Representative colonies from each plate were sequenced to identify the putative suppressor mutation. (B) Cartoon of the cohesin complex showing relative positions of the indicated smc3-K113Q suppressor mutations. (Cyan) Smc1p, (purple) Smc3p, (green) Mcd1p. Also depicted is cohesin-associating protein Scc2p (salmon). (C) Residues of three suppressors—Smc1p-R1199, Smc1p-T1117, and Smc1p-A159—map close to the Smc3p ATPase and Scc2p. Cryo-EM structure of the S. cerevisiae (PDB ID: 6ZZ6) (Collier et al. 2020) Smc1p (cyan) and Smc3p (magenta) head domains bound to Scc2p (salmon). The S. cerevisiae Smc3p-R1199 (magenta spheres), S. cerevisiae Smc1p-T1117 (cyan spheres), and S. cerevisiae Smc1p-A159 (cyan spheres) residues are indicated. ATP (yellow) and each SMC ATPase are also indicated.

Figure 3.

Smc1-T1117I uniquely and robustly suppresses smc3-K113Q defects in viability, drug sensitivity, cohesion, and cohesin binding to DNA. The smc1-T1117I mutant is a robust suppressor of the smc3-K113Q mutant. (A) The smc1-T1117I smc3-K113Q double mutant grows as well as WT and is resistant to drugs. The haploid wild-type (VG4012-2C), smc1-T1117I (VG4006-13A), and smc3-K113Q smc1-T1117I (VG4010-8B) strains were grown and diluted as described in Figure 1C; plated on YPD alone or containing 10 μg/mL benomyl (BEN) or 15 μg/mL camptothecin (CPT); and incubated for 3 d at 23°C, 4 d at 23°C, or 3 d at 30°C, respectively. Plates were electronically rearranged for ease of display. (B) smc1-T1117I strongly suppresses the cohesion defect of the smc3-K113Q mutant. The haploid wild-type (VG3620-4C), smc3-K113Q smc3-AID double-mutant (VG3891-6B), smc1-T1117I (VG4006-13A), and smc3-K113Q smc1-T1117I double-mutant (VG4010-8B) cells were arrested in G1; auxin was added to deplete Smc3p-AID; and cells were synchronously released from G1 and arrested in mid-M phase. Cohesion loss at a chromosome IV arm locus was assessed and plotted as described in Figure 1D. (C) The smc1-T1117I smc3-K113Q double-mutant cohesin binds to chromosomes at levels equal to or higher than wild-type cohesin. Mid-M-phase cells from B were fixed and processed for ChIP, and the level of cohesin bound to chromosomes was determined as described in Figure 1E. (Left panel) Chromosome IV arm CAR region (TRM1). (Middle panel) Chromosome III pericentric region (CARC1). (Right panel) Regions immediately flanking CEN14 and CEN4.

Figure 4.

Model for Scc2p-mediated stimulation of Smc3p ATPase and its inhibition by acetylation. (A) Cryo-EM structure of the S. cerevisiae cohesin (PDB ID: 6ZZ6) (Collier et al. 2020) interface between Smc1p (cyan) and Scc2p (salmon), indicating Smc1p-T1117, Smc1p-K1121, and ATP (yellow). (B) Cartoon of the cohesin head domain in its basal ATPase state, with key Smc1p and Smc3p residues indicated. (C) Cartoon of the cohesin head domain in its stimulated ATPase state with Scc2p bound when Smc3p in not acetylated (left) and the inhibited state with Smc3p-K113 acetylated (right). (Left) Scc2p stimulation of cohesin ATPase: (1) Scc2p-E822 is in close proximity to unacetylated Smc3p-K113, which (2) properly orients the Scc2p–Smc1p interface. Scc2p binding at this Smc1p interface (3) shifts Smc1p and ATP such that (4) ATP is nearer the Smc3p-E1155 catalytic glutamate. (Right) Acetylated cohesin inhibits Scc2p stimulation of cohesin ATPase: (1) Acetylation of Smc3p-K113 disrupts Scc2p-E822 positioning, leading to (2) improper binding of Scc2p at the Smc1p-T1117 interface, resulting in (3) a failure to reposition the ATP closer to the catalytic glutamate, thereby (4) inhibiting Scc2p's stimulation of cohesin's ATPase.

Figure 5.

Only isoleucine or valine residue substitutions at smc1-T1117 suppress smc3-K113Q. (A) Schematic of a screen to assess which substitutions at smc1-T1117 suppress smc3-K113Q. The haploid strain (VG3969-14C) contains smc3-K113Q as the sole S. cerevisiae SMC3 and S. Bayanus SMC3 on a CEN URA3 G418 plasmid (pFC3). CRISPR was used to insert random substitutions of the smc1-T1117 residues as described in the Materials and Methods. Transformants were replica-plated to FOA media to select for loss of pFC3 (FOA^R G418^S) colonies, which were sequenced to identify smc1-T1117 substitutions that suppress smc3-K113Q inviability. (B) The valine substitution at smc1-T1117 (T1117V) is a weaker suppressor of smc3-K113Q than isoleucine (T1117I). Wild-type (VG3620-4C), smc1-T1117I (VG4006-13A), smc3-K113Q smc1-T1117I (VG4010-8B), and smc3-K113Q smc1-T1117V (VG4147-14C) were grown and diluted as described in Figure 1C and plated on YPD and incubated for 4 d at 23°C or for 3 d at 30°C and 37°C or plated on YPD containing 10 μg/mL benomyl (BEN) or 15 μg/mL camptothecin (CPT) and incubated for 4 d at 23°C. (C) Model generated from the cryo-EM structure of S. cerevisiae cohesin (PDB ID: 6ZZ6) (Collier et al. 2020) depicting the interface between Smc1p-T1117 (cyan) and Scc2p (salmon). (Left) The interface between Scc2p and wild-type Smc1p. Structure models of Smc3p-K113Q suppressors Smc1p-T1117V (middle) and Smc1p-T1117I (right) are also shown.

Figure 6.

A tryptophan substitution at smc1-T1117 (T1117W) strongly suppresses the smc3-K112R, K113R (acetyl-null) mutant. (A) smc1-T1117I exacerbates the growth defect of ECO1-AID depletion. Wild-type (VG3620-4C), ECO1-AID (VG3633-3D), smc1-T1117I ECO1-AID (KB118E), and smc1-T1117I (JL11A) strains were grown and diluted as in Figure 1C, plated on YPD and YPD + IAA, and incubated for 5 d at 23°C. (B) smc1-T1117W and smc1-D1164E strongly suppress smc3-K112R, K113R (RR). The haploid wild-type (VG3620-4C); wpl1Δ smc3-K112R, K113R (VG4154-3A); smc1-D1164E smc3-K112R, K113R (VG4153-5C); and smc1-T1117W smc3-K112R, K113R (VG4158-9D) strains were grown and diluted as in Figure 1C and plated on YPD and incubated for 4 d at 23°C, for 3 d at 30°C, or for 3 d at 37°C or plated on YPD containing 10 μg/mL benomyl and incubated for 5 d at 23°C (BEN 23°C). (C) smc1-T1117W strongly suppresses cohesion defect of the smc3-K112R, K113R mutant. The haploid wild-type (VG3620-4C); smc3-AID smc3-K112R, K113R (VG3991-1A); smc1-T1117W (VG4168-7B); smc1-T1117W smc3-K112R, K113R (VG4158-9D); smc1-D1164E (VG4138-1A); and smc1-D1164E smc3-K112R, K113R (VG4153-5C) strains were grown as described in Figure 3B and processed to assess cohesion loss as described in Figure 1D. (D) Model generated from the cryo-EM structure of S. cerevisiae cohesin (PDB ID: 6ZZ6) (Collier et al. 2020) illustrating wild-type Smc1p-T1117 (left) and acetyl-defective suppressor Smc1p-T1117W substitution (right).

Figure 7.

Different substitutions at SMC1-T1117 up-regulate or down-regulate cohesin ATPase activity. (A) The Smc1p-T1117I suppressor of the Smc3p acetyl-mimic up-regulates cohesin ATPase activity. Wild-type and mutant cohesin complexes were purified and assessed for ATPase activity in the presence of DNA with and without Scc2p/Scc4p (loader). Purified cohesin concentration normalization was confirmed (Supplemental Fig. S6A). (B) The Smc1p-T1117W and Smc1p-D1164E suppressors of the Smc3 acetyl-null down-regulate cohesin ATPase activity. Wild-type and mutant cohesin complexes were purified and assessed for ATPase activity in the presence of DNA with and without Scc2p/Scc4p (loader). Purified cohesin concentration normalization was confirmed (Supplemental Fig. S6B).