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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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ATPase mutant causes mitotic phenotype, and protein thiophosphorylation can occur in terminally truncated SMC proteins when one of two SMCs is intact. (a) Plasmid pREP1 carrying the Cut14 G35N mutant gene under the inducible promoter was used to overexpress it simultaneously with the wild-type cut3+ gene (plasmid pREP2) in the absence of thiamine (−Thi). See text. (b) Cells were stained by DAPI. In liquid culture, a defective phenotype in chromosome condensation and segregation was observed after co-overexpressing (OP) the wild-type Cut3 and the ATPase mutant Cut14 G35N (right panel). See text. Scale bars, 10 µm. (c) Cut3–Cut14 complex was incubated with γP32-ATP in the presence or the absence of M13 ssDNA. The autoradiography pattern (P32) and CBB staining patterns are shown. (d) Antibody against thiophosphorylated amino acids (α-ATPγS) was used to detect thiophosphorylated residues of Cut3–Cut14 incubated in the presence of ATPγS, hdDNA and PNBM (the reagent for detecting γS). The Cut3 band containing γS was detected when Cut3–Cut14 was incubated with ATPγS and PNBM, regardless of the presence of hdDNA. Protein bands of Cut3 and Cut14-3HA6His were stained with Ponceau S. (e) Wild-type Cut3/SMC4 and Cut14/SMC2, and three single- or double-truncated Cut3 and wild-type Cut14 constructs are shown. (f) Wild-type and truncated SMC dimer were incubated with γP32-ATP, run in SDS-PAGE, and examined by autoradiography of labelled proteins (i) and CBB staining (ii). Red arrows indicate bands of Cut3 wild-type and mutant truncated proteins in autoradiography and CBB stain.
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RSOB140193F2: ATPase mutant causes mitotic phenotype, and protein thiophosphorylation can occur in terminally truncated SMC proteins when one of two SMCs is intact. (a) Plasmid pREP1 carrying the Cut14 G35N mutant gene under the inducible promoter was used to overexpress it simultaneously with the wild-type cut3+ gene (plasmid pREP2) in the absence of thiamine (−Thi). See text. (b) Cells were stained by DAPI. In liquid culture, a defective phenotype in chromosome condensation and segregation was observed after co-overexpressing (OP) the wild-type Cut3 and the ATPase mutant Cut14 G35N (right panel). See text. Scale bars, 10 µm. (c) Cut3–Cut14 complex was incubated with γP32-ATP in the presence or the absence of M13 ssDNA. The autoradiography pattern (P32) and CBB staining patterns are shown. (d) Antibody against thiophosphorylated amino acids (α-ATPγS) was used to detect thiophosphorylated residues of Cut3–Cut14 incubated in the presence of ATPγS, hdDNA and PNBM (the reagent for detecting γS). The Cut3 band containing γS was detected when Cut3–Cut14 was incubated with ATPγS and PNBM, regardless of the presence of hdDNA. Protein bands of Cut3 and Cut14-3HA6His were stained with Ponceau S. (e) Wild-type Cut3/SMC4 and Cut14/SMC2, and three single- or double-truncated Cut3 and wild-type Cut14 constructs are shown. (f) Wild-type and truncated SMC dimer were incubated with γP32-ATP, run in SDS-PAGE, and examined by autoradiography of labelled proteins (i) and CBB staining (ii). Red arrows indicate bands of Cut3 wild-type and mutant truncated proteins in autoradiography and CBB stain.

Mentions: We examined whether abnormal cellular phenotypes resulted from overproduction by plasmids carrying the ATPase G35N substitution mutant gene. The cut14 G35N substitution mutant gene was overexpressed under an inducible promoter, using plasmid pREP1, with co-overexpression of the wild-type cut3+ gene carried by plasmid pREP2 in the absence of thiamine (−Thi) so that wild-type Cut3 and mutant Cut14 G35N were overproduced, forming the heterodimer and causing a dominant-negative effect (no colony formed; figure 2a, right bottom). Under conditions to suppress overexpression (+Thi), S. pombe cells carrying the mutant plasmid formed normal colonies (figure 2a, right top). Thus, co-overexpression (−Thi) of Cut14 G35N and wild-type Cut3 proteins was strongly inhibitory.Figure 2.


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)

ATPase mutant causes mitotic phenotype, and protein thiophosphorylation can occur in terminally truncated SMC proteins when one of two SMCs is intact. (a) Plasmid pREP1 carrying the Cut14 G35N mutant gene under the inducible promoter was used to overexpress it simultaneously with the wild-type cut3+ gene (plasmid pREP2) in the absence of thiamine (−Thi). See text. (b) Cells were stained by DAPI. In liquid culture, a defective phenotype in chromosome condensation and segregation was observed after co-overexpressing (OP) the wild-type Cut3 and the ATPase mutant Cut14 G35N (right panel). See text. Scale bars, 10 µm. (c) Cut3–Cut14 complex was incubated with γP32-ATP in the presence or the absence of M13 ssDNA. The autoradiography pattern (P32) and CBB staining patterns are shown. (d) Antibody against thiophosphorylated amino acids (α-ATPγS) was used to detect thiophosphorylated residues of Cut3–Cut14 incubated in the presence of ATPγS, hdDNA and PNBM (the reagent for detecting γS). The Cut3 band containing γS was detected when Cut3–Cut14 was incubated with ATPγS and PNBM, regardless of the presence of hdDNA. Protein bands of Cut3 and Cut14-3HA6His were stained with Ponceau S. (e) Wild-type Cut3/SMC4 and Cut14/SMC2, and three single- or double-truncated Cut3 and wild-type Cut14 constructs are shown. (f) Wild-type and truncated SMC dimer were incubated with γP32-ATP, run in SDS-PAGE, and examined by autoradiography of labelled proteins (i) and CBB staining (ii). Red arrows indicate bands of Cut3 wild-type and mutant truncated proteins in autoradiography and CBB stain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140193F2: ATPase mutant causes mitotic phenotype, and protein thiophosphorylation can occur in terminally truncated SMC proteins when one of two SMCs is intact. (a) Plasmid pREP1 carrying the Cut14 G35N mutant gene under the inducible promoter was used to overexpress it simultaneously with the wild-type cut3+ gene (plasmid pREP2) in the absence of thiamine (−Thi). See text. (b) Cells were stained by DAPI. In liquid culture, a defective phenotype in chromosome condensation and segregation was observed after co-overexpressing (OP) the wild-type Cut3 and the ATPase mutant Cut14 G35N (right panel). See text. Scale bars, 10 µm. (c) Cut3–Cut14 complex was incubated with γP32-ATP in the presence or the absence of M13 ssDNA. The autoradiography pattern (P32) and CBB staining patterns are shown. (d) Antibody against thiophosphorylated amino acids (α-ATPγS) was used to detect thiophosphorylated residues of Cut3–Cut14 incubated in the presence of ATPγS, hdDNA and PNBM (the reagent for detecting γS). The Cut3 band containing γS was detected when Cut3–Cut14 was incubated with ATPγS and PNBM, regardless of the presence of hdDNA. Protein bands of Cut3 and Cut14-3HA6His were stained with Ponceau S. (e) Wild-type Cut3/SMC4 and Cut14/SMC2, and three single- or double-truncated Cut3 and wild-type Cut14 constructs are shown. (f) Wild-type and truncated SMC dimer were incubated with γP32-ATP, run in SDS-PAGE, and examined by autoradiography of labelled proteins (i) and CBB staining (ii). Red arrows indicate bands of Cut3 wild-type and mutant truncated proteins in autoradiography and CBB stain.
Mentions: We examined whether abnormal cellular phenotypes resulted from overproduction by plasmids carrying the ATPase G35N substitution mutant gene. The cut14 G35N substitution mutant gene was overexpressed under an inducible promoter, using plasmid pREP1, with co-overexpression of the wild-type cut3+ gene carried by plasmid pREP2 in the absence of thiamine (−Thi) so that wild-type Cut3 and mutant Cut14 G35N were overproduced, forming the heterodimer and causing a dominant-negative effect (no colony formed; figure 2a, right bottom). Under conditions to suppress overexpression (+Thi), S. pombe cells carrying the mutant plasmid formed normal colonies (figure 2a, right top). Thus, co-overexpression (−Thi) of Cut14 G35N and wild-type Cut3 proteins was strongly inhibitory.Figure 2.

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