PICH promotes SUMOylated TopoisomeraseIIα dissociation from mitotic centromeres for proper chromosome segregation

AbstractProper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by abnormal distribution of genetic information. Polo-like kinase 1 interacting checkpoint helicase (PICH) is a DNA translocase essential in chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by Small Ubiquitin-like modifier (SUMO). However, it is unclear how these activities are cooperating to resolve chromosome bridges. Here, we show that PICH specifically promotes the organization of SUMOylated proteins like SUMOylated TopoisomeraseIIα (TopoIIα) on mitotic chromosomes. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in the retention of SUMOylated chromosomal proteins, including TopoIIα, indicating that PICH functions to control proper association of these proteins with chromosomes. Replacement of PICH with its mutants showed that PICH is required for the proper organization of SUMOylated proteins on chromosomes. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taken together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.Summary StatementPolo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH controls association of SUMOylated chromosomal proteins, including Topoisomerase IIα, and that function requires PICH translocase activity and SUMO binding ability.

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An essential yeast gene encoding a TTAGGG repeat-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1306-1314.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1306-1314

Author(s):

C Brigati ◽

S Kurtz ◽

D Balderes ◽

G Vidali ◽

D Shore

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Binding Activity ◽

Insertion Mutation ◽

Yeast Gene ◽

Gene Encoding ◽

Cloned Gene ◽

Diploid Cells ◽

Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

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The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3419 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3419-3424 ◽

Cited By ~ 130

Author(s):

C G Burd ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Consensus Sequence ◽

Binding Activity ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal ◽

Binding Domains ◽

Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

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Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

Eukaryotic Cell ◽

10.1128/ec.3.5.1185-1197.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1185-1197 ◽

Cited By ~ 22

Author(s):

Bidyottam Mittra ◽

Dan S. Ray

Keyword(s):

Cell Cycle ◽

Binding Protein ◽

High Specificity ◽

Binding Activity ◽

Mrna Levels ◽

Crithidia Fasciculata ◽

Target Mrna ◽

Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

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Simple topology: FtsK-directed recombination at the dif site

Biochemical Society Transactions ◽

10.1042/bst20120299 ◽

2013 ◽

Vol 41 (2) ◽

pp. 595-600 ◽

Cited By ~ 11

Author(s):

Ian Grainge

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Chromosome Segregation ◽

Motor Domain ◽

Site Specific ◽

Multifunctional Protein ◽

Supercoiled Plasmid ◽

C Terminus ◽

Dna Translocase

FtsK is a multifunctional protein, which, in Escherichia coli, co-ordinates the essential functions of cell division, DNA unlinking and chromosome segregation. Its C-terminus is a DNA translocase, the fastest yet characterized, which acts as a septum-localized DNA pump. FtsK's C-terminus also interacts with the XerCD site-specific recombinases which act at the dif site, located in the terminus region. The motor domain of FtsK is an active translocase in vitro, and, when incubated with XerCD and a supercoiled plasmid containing two dif sites, recombination occurs to give unlinked circular products. Despite years of research the mechanism for this novel form of topological filter remains unknown.

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A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

The Journal of Cell Biology ◽

10.1083/jcb.113.6.1243 ◽

1991 ◽

Vol 113 (6) ◽

pp. 1243-1254 ◽

Cited By ~ 28

Author(s):

U Stochaj ◽

M Osborne ◽

T Kurihara ◽

P Silver

Keyword(s):

Nuclear Localization ◽

Binding Proteins ◽

Protein Import ◽

Nuclear Protein ◽

Binding Protein ◽

Protein Localization ◽

Binding Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Nuclear Localization Signals

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

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DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1671 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1671-1685 ◽

Cited By ~ 83

Author(s):

Mohammed Momin Shareef ◽

Chadwick King ◽

Mona Damaj ◽

RamaKrishna Badagu ◽

Da Wei Huang ◽

...

Keyword(s):

Dna Binding ◽

Origin Recognition Complex ◽

Polytene Chromosomes ◽

Binding Activity ◽

Hmg Proteins ◽

Mitotic Chromosomes ◽

Origin Firing ◽

Dna Binding Activity ◽

Origin Recognition

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

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Changes in cytosolic 3,5,3′-tri-iodo-l-thyronine (T3) binding activity during administration of l-thyroxine to thyroidectomized rats: cytosolic T3-binding protein and its activator act as intracellular regulators for nuclear T3 binding

Journal of Endocrinology ◽

10.1677/joe.0.1230099 ◽

1989 ◽

Vol 123 (1) ◽

pp. 99-104 ◽

Cited By ~ 7

Author(s):

Y. Nishii ◽

K. Hashizume ◽

K. Ichikawa ◽

T. Miyamoto ◽

S. Suzuki ◽

...

Keyword(s):

Thyroid Hormone ◽

Nuclear Receptor ◽

Binding Capacity ◽

Binding Protein ◽

Binding Activity ◽

Affinity Constant ◽

Maximal Binding

ABSTRACT Changes in the amount of cytosolic 3,5,3′-tri-iodo-l-thyronine (T3)-binding protein (CTBP) and its activator during administration of l-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor. These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor. Journal of Endocrinology (1989) 123, 99–104

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An essential yeast gene encoding a TTAGGG repeat-binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1306 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1306-1314 ◽

Cited By ~ 51

Author(s):

C Brigati ◽

S Kurtz ◽

D Balderes ◽

G Vidali ◽

D Shore

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Binding Activity ◽

Insertion Mutation ◽

Yeast Gene ◽

Gene Encoding ◽

Cloned Gene ◽

Diploid Cells ◽

Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

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Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

Eukaryotic Cell ◽

10.1128/ec.3.2.277-287.2004 ◽

2004 ◽

Vol 3 (2) ◽

pp. 277-287 ◽

Cited By ~ 46

Author(s):

Itay Onn ◽

Neta Milman-Shtepel ◽

Joseph Shlomai

Keyword(s):

Dna Binding ◽

Redox Potential ◽

Protein Interactions ◽

Replication Origin ◽

Binding Protein ◽

Binding Activity ◽

Kinetoplast Dna ◽

Minicircle Sequence

ABSTRACT Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

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