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ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

Akai Y, Kanai R, Nakazawa N, Ebe M, Toyoshima C, Yanagida M - Open Biol (2014)

Bottom Line: Phosphorylation reduces affinity for DNA.Consistently, phosphomimetic mutants produce severe mitotic phenotypes.Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan.

ABSTRACT
Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

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Related in: MedlinePlus

(Thio)Phosphorylation sites of Cut3/SMC4 are located in the G-rich sequence along the hinge dimer interface. (a) Purified Cut3–Cut14 was incubated without ATP, or in the presence of ATP or ATPγS, and reisolated for LC-MS analysis. Phospho- or thiophosphopeptides of Cut3 are shown with the amino acid sequence number and the sequences containing phosphorylated (blue) and thiophosphorylated (red) residues (also shown in electronic supplementary material, tables S1 and S2). The majority of residues are located in the hinge (616–798) and hinge-adjacent regions. (b) Schematic of Cut3 protein with thiophosphorylated residues (red). The Walker motif sequences shown by the vertical bars (black) are located in the N- and C-termini. (c) An expected structure of the heterodimeric hinge of Cut3/SMC4 (yellow) and Cut14/SMC2 (green). Thiophosphorylated residues are located along the interface of the dimer. T785 and T787 are located in a β-strand (β7, see text). The asterisk indicates G626 at the base of helix H that works as a pivot in opening/closing of the Cut14/SMC2 hinge (see Discussion). (d) Top panel: locations of the thiophosphorylated residues (red discs labelled P) and the surrounding basic residues (‘+’) in the hinge doughnut (see text). Arrows indicate the β-strands. Bottom: details around interface 1. Thiophosphorylated residues are coloured green (S630 and S633), orange (T774) or magenta (S783, T785, T787 and T791). Surrounding basic residues appear in blue and are labelled with normal letters for Cut3/SMC4 and italics for Cut14/SMC2. Hydrogen bonds are indicated by dotted lines. Basic residues of Cut14/SMC2 are shown in italics. Basic residues of Cut3/SMC4 are also seen. (e) cut3-477 mutant cells were transformed with plasmids carrying no gene (vector), wild-type cut3+ (WT) and the cut3 mutant gene containing 5A or 5E substitutions (see text), and resulting transformants were spot tested at 26°C and 36°C in the presence of thiamine (induction is off). (f) Immunological detection of the phosphorylated Cut3-T787-P site. Cut3 protein was isolated from cells that overproduced Cut3-3HA6His, incubated in the absence of ATP, or the presence of 0.5 and 1.0 mM ATP, and run on SDS-PAGE. An antibody raised against the peptide KSGTMT787PGGGTRYK containing phospho T787 was used for detection.
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RSOB140193F3: (Thio)Phosphorylation sites of Cut3/SMC4 are located in the G-rich sequence along the hinge dimer interface. (a) Purified Cut3–Cut14 was incubated without ATP, or in the presence of ATP or ATPγS, and reisolated for LC-MS analysis. Phospho- or thiophosphopeptides of Cut3 are shown with the amino acid sequence number and the sequences containing phosphorylated (blue) and thiophosphorylated (red) residues (also shown in electronic supplementary material, tables S1 and S2). The majority of residues are located in the hinge (616–798) and hinge-adjacent regions. (b) Schematic of Cut3 protein with thiophosphorylated residues (red). The Walker motif sequences shown by the vertical bars (black) are located in the N- and C-termini. (c) An expected structure of the heterodimeric hinge of Cut3/SMC4 (yellow) and Cut14/SMC2 (green). Thiophosphorylated residues are located along the interface of the dimer. T785 and T787 are located in a β-strand (β7, see text). The asterisk indicates G626 at the base of helix H that works as a pivot in opening/closing of the Cut14/SMC2 hinge (see Discussion). (d) Top panel: locations of the thiophosphorylated residues (red discs labelled P) and the surrounding basic residues (‘+’) in the hinge doughnut (see text). Arrows indicate the β-strands. Bottom: details around interface 1. Thiophosphorylated residues are coloured green (S630 and S633), orange (T774) or magenta (S783, T785, T787 and T791). Surrounding basic residues appear in blue and are labelled with normal letters for Cut3/SMC4 and italics for Cut14/SMC2. Hydrogen bonds are indicated by dotted lines. Basic residues of Cut14/SMC2 are shown in italics. Basic residues of Cut3/SMC4 are also seen. (e) cut3-477 mutant cells were transformed with plasmids carrying no gene (vector), wild-type cut3+ (WT) and the cut3 mutant gene containing 5A or 5E substitutions (see text), and resulting transformants were spot tested at 26°C and 36°C in the presence of thiamine (induction is off). (f) Immunological detection of the phosphorylated Cut3-T787-P site. Cut3 protein was isolated from cells that overproduced Cut3-3HA6His, incubated in the absence of ATP, or the presence of 0.5 and 1.0 mM ATP, and run on SDS-PAGE. An antibody raised against the peptide KSGTMT787PGGGTRYK containing phospho T787 was used for detection.

Mentions: For mass spectrometric analysis, we purified Cut3ΔNC–Cut14 complex (electronic supplementary material, figure S2), which was incubated in control buffer (no ATP, lane 1) in the presence of ATP (ATP, lane 2) and in the presence of ATPγS (ATPγS, lane 3) at 30°C for 90 min. CBB staining is also shown. Slices of the SDS-PAGE gel were digested with trypsin or lysine-specific Lys-C protease, and resulting digests were analysed by mass spectrometry as described previously [18]. Identified phosphorylated or thiophosphorylated peptides, with ion score values (obtained with Mascot software for Orbitrap) or the number of peptides, respectively, are shown in the electronic supplementary material, tables S1 (trypsin digestion) and S2 (Lys-C digestion). These results are summarized in figure 3a,b.Figure 3.


ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

Akai Y, Kanai R, Nakazawa N, Ebe M, Toyoshima C, Yanagida M - Open Biol (2014)

(Thio)Phosphorylation sites of Cut3/SMC4 are located in the G-rich sequence along the hinge dimer interface. (a) Purified Cut3–Cut14 was incubated without ATP, or in the presence of ATP or ATPγS, and reisolated for LC-MS analysis. Phospho- or thiophosphopeptides of Cut3 are shown with the amino acid sequence number and the sequences containing phosphorylated (blue) and thiophosphorylated (red) residues (also shown in electronic supplementary material, tables S1 and S2). The majority of residues are located in the hinge (616–798) and hinge-adjacent regions. (b) Schematic of Cut3 protein with thiophosphorylated residues (red). The Walker motif sequences shown by the vertical bars (black) are located in the N- and C-termini. (c) An expected structure of the heterodimeric hinge of Cut3/SMC4 (yellow) and Cut14/SMC2 (green). Thiophosphorylated residues are located along the interface of the dimer. T785 and T787 are located in a β-strand (β7, see text). The asterisk indicates G626 at the base of helix H that works as a pivot in opening/closing of the Cut14/SMC2 hinge (see Discussion). (d) Top panel: locations of the thiophosphorylated residues (red discs labelled P) and the surrounding basic residues (‘+’) in the hinge doughnut (see text). Arrows indicate the β-strands. Bottom: details around interface 1. Thiophosphorylated residues are coloured green (S630 and S633), orange (T774) or magenta (S783, T785, T787 and T791). Surrounding basic residues appear in blue and are labelled with normal letters for Cut3/SMC4 and italics for Cut14/SMC2. Hydrogen bonds are indicated by dotted lines. Basic residues of Cut14/SMC2 are shown in italics. Basic residues of Cut3/SMC4 are also seen. (e) cut3-477 mutant cells were transformed with plasmids carrying no gene (vector), wild-type cut3+ (WT) and the cut3 mutant gene containing 5A or 5E substitutions (see text), and resulting transformants were spot tested at 26°C and 36°C in the presence of thiamine (induction is off). (f) Immunological detection of the phosphorylated Cut3-T787-P site. Cut3 protein was isolated from cells that overproduced Cut3-3HA6His, incubated in the absence of ATP, or the presence of 0.5 and 1.0 mM ATP, and run on SDS-PAGE. An antibody raised against the peptide KSGTMT787PGGGTRYK containing phospho T787 was used for detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4281712&req=5

RSOB140193F3: (Thio)Phosphorylation sites of Cut3/SMC4 are located in the G-rich sequence along the hinge dimer interface. (a) Purified Cut3–Cut14 was incubated without ATP, or in the presence of ATP or ATPγS, and reisolated for LC-MS analysis. Phospho- or thiophosphopeptides of Cut3 are shown with the amino acid sequence number and the sequences containing phosphorylated (blue) and thiophosphorylated (red) residues (also shown in electronic supplementary material, tables S1 and S2). The majority of residues are located in the hinge (616–798) and hinge-adjacent regions. (b) Schematic of Cut3 protein with thiophosphorylated residues (red). The Walker motif sequences shown by the vertical bars (black) are located in the N- and C-termini. (c) An expected structure of the heterodimeric hinge of Cut3/SMC4 (yellow) and Cut14/SMC2 (green). Thiophosphorylated residues are located along the interface of the dimer. T785 and T787 are located in a β-strand (β7, see text). The asterisk indicates G626 at the base of helix H that works as a pivot in opening/closing of the Cut14/SMC2 hinge (see Discussion). (d) Top panel: locations of the thiophosphorylated residues (red discs labelled P) and the surrounding basic residues (‘+’) in the hinge doughnut (see text). Arrows indicate the β-strands. Bottom: details around interface 1. Thiophosphorylated residues are coloured green (S630 and S633), orange (T774) or magenta (S783, T785, T787 and T791). Surrounding basic residues appear in blue and are labelled with normal letters for Cut3/SMC4 and italics for Cut14/SMC2. Hydrogen bonds are indicated by dotted lines. Basic residues of Cut14/SMC2 are shown in italics. Basic residues of Cut3/SMC4 are also seen. (e) cut3-477 mutant cells were transformed with plasmids carrying no gene (vector), wild-type cut3+ (WT) and the cut3 mutant gene containing 5A or 5E substitutions (see text), and resulting transformants were spot tested at 26°C and 36°C in the presence of thiamine (induction is off). (f) Immunological detection of the phosphorylated Cut3-T787-P site. Cut3 protein was isolated from cells that overproduced Cut3-3HA6His, incubated in the absence of ATP, or the presence of 0.5 and 1.0 mM ATP, and run on SDS-PAGE. An antibody raised against the peptide KSGTMT787PGGGTRYK containing phospho T787 was used for detection.
Mentions: For mass spectrometric analysis, we purified Cut3ΔNC–Cut14 complex (electronic supplementary material, figure S2), which was incubated in control buffer (no ATP, lane 1) in the presence of ATP (ATP, lane 2) and in the presence of ATPγS (ATPγS, lane 3) at 30°C for 90 min. CBB staining is also shown. Slices of the SDS-PAGE gel were digested with trypsin or lysine-specific Lys-C protease, and resulting digests were analysed by mass spectrometry as described previously [18]. Identified phosphorylated or thiophosphorylated peptides, with ion score values (obtained with Mascot software for Orbitrap) or the number of peptides, respectively, are shown in the electronic supplementary material, tables S1 (trypsin digestion) and S2 (Lys-C digestion). These results are summarized in figure 3a,b.Figure 3.

Bottom Line: Phosphorylation reduces affinity for DNA.Consistently, phosphomimetic mutants produce severe mitotic phenotypes.Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan.

ABSTRACT
Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

Show MeSH
Related in: MedlinePlus