23 results on '"Takahisa Maki"'
Search Results
2. Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex
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Bilge Argunhan, Masayoshi Sakakura, Negar Afshar, Misato Kurihara, Kentaro Ito, Takahisa Maki, Shuji Kanamaru, Yasuto Murayama, Hideo Tsubouchi, Masayuki Takahashi, Hideo Takahashi, and Hiroshi Iwasaki
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DNA repair ,homologous recombination ,Rad51 ,Swi5-Sfr1 ,Rad55-Rad57 ,disordered ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.
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- 2020
- Full Text
- View/download PDF
3. New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe.
- Author
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Takahisa Maki, Naoto Ogura, James E Haber, Hiroshi Iwasaki, and Geneviève Thon
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Genetics ,QH426-470 - Abstract
Mating-type switching in Schizosaccharomyces pombe entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, mat2-P or mat3-M, is chosen to convert the expressed mat1 locus. By measuring the biases in mat1 content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (mat2-M mat3-P) or when the SRE2 and SRE3 recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from mat2 and the SRE2 enhancer, towards mat3 and the SRE3 enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD+-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD+-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to mat2-P in M cells, perhaps by inhibiting the SRE3 recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.
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- 2018
- Full Text
- View/download PDF
4. Fission yeast Swi2 designates cell-type specific donor and stimulates Rad51-driven strand exchange for mating-type switching
- Author
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Takahisa Maki, Geneviève Thon, and Hiroshi Iwasaki
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EXPRESSION ,HOMOLOG ,SCHIZOSACCHAROMYCES-POMBE ,SWI5-SFR1 COMPLEX ,PROTEINS ,DNA-REPLICATION ,Genetics ,RECOMBINATION ,DIRECTIONALITY ,IMPRINT ,CENP-B - Abstract
A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M mating-type, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis-acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site. A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M mating-type, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis-acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.
- Published
- 2023
5. Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe
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Alfredo Esquivel-Chávez, Takahisa Maki, Hideo Tsubouchi, Testuya Handa, Hiroshi Kimura, James E. Haber, Geneviève Thon, and Hiroshi Iwasaki
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Genetics ,General Medicine ,Molecular Biology - Published
- 2022
6. Fission yeast Swi2 designates cell-type specific donor and stimulates Rad51-driven strand exchange
- Author
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Takahisa Maki, Geneviève Thon, and Hiroshi Iwasaki
- Abstract
A haploid of the fission yeastSchizosaccharomyces pombeexpresses either the P or M matingtype, determined by the active, euchromatic,mat1cassette. Mating-type is switched by Rad51-driven gene conversion ofmat1using a heterochromatic donor cassette,mat2-Pormat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of twocisacting recombination enhancers,SRE2adjacent tomat2-PorSRE3adjacent tomat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization atSRE3to select themat3-Mdonor in P cells, while the Swi6-binding site was required for Swi2 localization atSRE2to selectmat2-Pin M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchangein vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.
- Published
- 2022
7. Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51
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Hideo Tsubouchi, Kentaro Ito, Mitsunori Ikeguchi, Tsutomu Mikawa, Masayuki Takahashi, Yumiko Kurokawa, Takahisa Maki, Bilge Argunhan, Yasuto Murayama, Yuichi Kokabu, Hiroshi Iwasaki, and Shuji Kanamaru
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0301 basic medicine ,DNA Repair ,DNA recombination ,Science ,genetic processes ,RAD51 ,General Physics and Astronomy ,DNA, Single-Stranded ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Adenosine Triphosphate ,Complementary DNA ,Schizosaccharomyces ,Recombinase ,Humans ,Homologous Recombination ,lcsh:Science ,Multidisciplinary ,Binding Sites ,Chemistry ,DNA damage and repair ,Nucleic Acid Heteroduplexes ,General Chemistry ,DNA ,DNA binding site ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Enzyme mechanisms ,Mutation ,Biophysics ,health occupations ,lcsh:Q ,Rad51 Recombinase ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,030217 neurology & neurosurgery ,Heteroduplex ,DNA Damage - Abstract
During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1–C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases., Rad51 drives DNA strand exchange, the central reaction in recombinational DNA repair. Two sites of Rad51 are responsible for DNA binding, but the function of these sites has proven elusive. Here, the authors employ real-time assays to reveal catalytic roles for the two DNA binding sites of Rad51.
- Published
- 2020
8. Mating-type switching by homology-directed recombinational repair: a matter of choice
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Takahisa Maki, Geneviève Thon, James E. Haber, and Hiroshi Iwasaki
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Homology-directed repair ,Heterochromatin ,Saccharomyces cerevisiae ,Review ,Chromatin structure ,Homology directed repair ,Histones ,03 medical and health sciences ,Yeasts ,Mating-type switching ,Schizosaccharomyces ,Genetics ,Homologous Recombination ,030304 developmental biology ,0303 health sciences ,biology ,Donor selection ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Histone modifications ,030302 biochemistry & molecular biology ,Recombinational DNA Repair ,General Medicine ,biology.organism_classification ,Genes, Mating Type, Fungal ,Chromatin ,Recombination ,Cell biology ,Mating of yeast ,Gene conversion ,Schizosaccharomyces pombe ,Mutation ,Homologous recombination - Abstract
In eukaryotes, all DNA transactions happen in the context of chromatin that often takes part in regulatory mechanisms. In particular, chromatin structure can regulate exchanges of DNA occurring through homologous recombination. Few systems have provided as detailed a view on this phenomenon as mating-type switching in yeast. Mating-type switching entails the choice of a template for the gene conversions of the expressed mating-type locus. In the fission yeast Schizosaccharomyces pombe, correct template choice requires two competing small recombination enhancers, SRE2 and SRE3, that function in the context of heterochromatin. These two enhancers act with the Swi2/Swi5 recombination accessory complex to initiate strand exchange in a cell-type-specific manner, from SRE2 in M cells and SRE3 in P cells. New research indicates that the Set1C complex, responsible for H3K4 methylation, and the Brl2 ubiquitin ligase, that catalyzes H2BK119 ubiquitylation, participate in the cell-type-specific selection of SRE2 or SRE3. Here, we review these findings, compare donor preference in S. pombe to the distantly related budding yeast Saccharomyces cerevisiae, and contrast the positive effects of heterochromatin on the donor selection process with other situations, where heterochromatin represses recombination.
- Published
- 2018
9. Euchromatin factors HULC and Set1C affect heterochromatin organization for mating-type switching in fission yeast Schizosaccharomyces pombe
- Author
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James E. Haber, Hiroshi Kimura, Alfredo Esquivel Chavez, Takahisa Maki, Hiroshi Iwasaki, Testuya Handa, Geneviève Thon, and Hideo Tsubouchi
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HULC ,Histone ,Euchromatin ,biology ,Donor selection ,Chemistry ,Ubiquitin ligase complex ,biology.protein ,Nucleosome ,Heterochromatin protein 1 ,Heterochromatin organization ,Cell biology - Abstract
Mating-type switching (MTS) in fission yeast Schizosaccharomyces pombe is a highly regulated gene conversion event. In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the use of two recombination enhancers, SRE2 promoting use of mat2-P and SRE3 promoting use of mat3-M. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in mutants further showed that the regulation might be by inhibiting use of the SRE3 enhancer in M cells, in favor of SRE2. Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain. Author Summary Mating-type switching in the fission yeast Schizosaccharomyces pombe occurs by gene conversion using a donor, mat2 or mat3 located in a heterochromatin region. Multiple studies have shown that donor selection is critically affected by heterochromatic factors. Here, we document the role of euchromatic factors, the histone H2BK119 ubiquitin ligase complex HULC and the H3K4 methyltransferase complex Set1C, in donor selection. Mutational analysis indicated that HULC and Set1C inhibit donor choice at the mat3 cis-acting recombination enhancer SRE3 in M cells, through concerted histone modifications by the two complexes. Linking this effect with heterochromatin, mutants in each complex, shf1Δ and set1Δ strains, exhibited decreased association of the HP1 heterochromatic factor Swi6 with the SRE2 and SRE3 recombination enhancers at the mat2 and mat3 silent loci in M cells. These joint effects by the two complexes on MTS were observed even though other aspects, the methylation state of histone H3K9 and effects on gene silencing differed between the shf1Δ and set1Δ strains. The results provide insight into the regulation of recombination by chromatin structure.
- Published
- 2021
10. A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity
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Aleksandar Zdravković, Hiroshi Iwasaki, Bilge Argunhan, James M. Daley, Masayuki Takahashi, Takahisa Maki, Patrick Sung, Tatsuya Niwa, Yasuto Murayama, Kentaro Ito, Arijit Dutta, Shuji Kanamaru, and Hideo Tsubouchi
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Endonuclease ,chemistry.chemical_compound ,Schizosaccharomyces ,DNA Breaks, Double-Stranded ,Amino Acid Sequence ,Phosphorylation ,Casein Kinase II ,Conserved Sequence ,chemistry.chemical_classification ,Multidisciplinary ,biology ,C-terminus ,Biological Sciences ,biology.organism_classification ,Cell biology ,Amino acid ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,chemistry ,C terminal peptide ,Schizosaccharomyces pombe ,biology.protein ,Schizosaccharomyces pombe Proteins ,Peptides ,Homologous recombination ,DNA - Abstract
The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.
- Published
- 2021
11. Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe.
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Esquivel-Chávez, Alfredo, Takahisa Maki, Hideo Tsubouchi, Testuya Handa, Hiroshi Kimura, Haber, James E., Thon, Geneviève, and Hiroshi Iwasaki
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SCHIZOSACCHAROMYCES pombe ,HETEROCHROMATIN ,EUCHROMATIN ,GENE conversion ,HISTONE methylation - Abstract
Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1?. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
12. Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex
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Hideo Tsubouchi, Masayuki Takahashi, Hideo Takahashi, Negar Afshar, Hiroshi Iwasaki, Yasuto Murayama, Bilge Argunhan, Misato Kurihara, Takahisa Maki, Kentaro Ito, Shuji Kanamaru, and Masayoshi Sakakura
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,DNA Repair ,QH301-705.5 ,DNA repair ,Science ,RAD51 ,Chemical biology ,homologous recombination ,Swi5-Sfr1 ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein Domains ,Biochemistry and Chemical Biology ,Gene Expression Regulation, Fungal ,Schizosaccharomyces ,Gene expression ,Escherichia coli ,disordered ,Biology (General) ,chemistry.chemical_classification ,General Immunology and Microbiology ,General Neuroscience ,General Medicine ,Chromosomes and Gene Expression ,Yeast ,Cell biology ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Enzyme ,chemistry ,Rad51 ,Medicine ,Rad51 Recombinase ,Schizosaccharomyces pombe Proteins ,Rad55-Rad57 ,Homologous recombination ,030217 neurology & neurosurgery ,DNA ,DNA Damage ,Protein Binding ,Research Article ,S. pombe - Abstract
Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe., eLife digest The DNA within cells contains the instructions necessary for life and it must be carefully maintained. DNA is constantly being damaged by radiation and other factors so cells have evolved an arsenal of mechanisms that repair this damage. An enzyme called Rad51 drives one such DNA repair process known as homologous recombination. A pair of regulatory proteins known as the Swi5-Sfr1 complex binds to Rad51 and activates it. The complex can be thought of as containing two modules with distinct roles: one comprising the first half of the Sfr1 protein and that is capable of binding to Rad51, and a second consisting of the rest of Sfr1 bound to Swi5, which is responsible for activating Rad51. Here, Argunhan, Sakakura et al. used genetic and biochemical approaches to study how this first module, known as “Sfr1N”, interacts with Rad51 in a microbe known as fission yeast. The experiments showed that both modules of Swi5-Sfr1 were important for Rad51 to drive homologous recombination. Swi5-Sfr1 complexes carrying mutations in the region of Sfr1N that binds to Rad51 were unable to activate Rad51 in a test tube. However, fission yeast cells containing the same mutations were able to repair their DNA without problems. This was due to the presence of another pair of proteins known as the Rad55-Rad57 complex that also bound to Swi5-Sfr1. The findings of Argunhan, Sakakura et al. suggest that the Swi5-Sfr1 and Rad55-Rad57 complexes work together to activate Rad51. Many genetically inherited diseases and cancers have been linked to mutations in DNA repair proteins. The fundamental mechanisms of DNA repair are very similar from yeast to humans and other animals, therefore, understanding the details of DNA repair in yeast may ultimately benefit human health in the future.
- Published
- 2020
13. Author response: Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex
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Takahisa Maki, Misato Kurihara, Masayuki Takahashi, Bilge Argunhan, Kentaro Ito, Negar Afshar, Yasuto Murayama, Hideo Takahashi, Shuji Kanamaru, Masayoshi Sakakura, Hideo Tsubouchi, and Hiroshi Iwasaki
- Subjects
Chemistry ,Factor (programming language) ,Stimulation ,Neuroscience ,computer ,computer.programming_language - Published
- 2020
14. Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange
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Yumiko Kurokawa, Hideo Tsubouchi, Masayuki Takahashi, Yasuto Murayama, Takahisa Maki, Bilge Argunhan, Shuji Kanamaru, Mitsunori Ikeguchi, Yuichi Kokabu, Hiroshi Iwasaki, and Kentaro Ito
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Protein filament ,chemistry.chemical_compound ,Chemistry ,Complementary DNA ,RAD51 ,Recombinase ,Biophysics ,Homologous recombination ,DNA ,Heteroduplex ,Nucleoprotein - Abstract
During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.
- Published
- 2019
- Full Text
- View/download PDF
15. Rad51 Interaction Analysis Reveals a Functional Interplay Among Recombination Auxiliary Factors
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Shuji Kanamaru, Misato Kurihara, Masayuki Takahashi, Kentaro Ito, Masayoshi Sakakura, Yasuto Murayama, Hideo Tsubouchi, Hideo Takahashi, Hiroshi Iwasaki, Negar Afshar, Takahisa Maki, and Bilge Argunhan
- Subjects
DNA repair ,genetic processes ,Mutant ,RAD51 ,Stimulation ,In vitro ,Cell biology ,enzymes and coenzymes (carbohydrates) ,chemistry.chemical_compound ,chemistry ,health occupations ,biological phenomena, cell phenomena, and immunity ,Homologous recombination ,DNA ,Recombination - Abstract
Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.
- Published
- 2019
16. New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe
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Naoto Ogura, Geneviève Thon, James E. Haber, Takahisa Maki, and Hiroshi Iwasaki
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0301 basic medicine ,Cancer Research ,lcsh:QH426-470 ,Heterochromatin ,DNA Mutational Analysis ,Gene Conversion ,Histones ,03 medical and health sciences ,Schizosaccharomyces ,Genetics ,Histone code ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,biology ,Methyltransferase complex ,Donor selection ,Genes, Mating Type, Fungal ,biology.organism_classification ,Cell biology ,Ubiquitin ligase ,Histone Code ,lcsh:Genetics ,030104 developmental biology ,Mating of yeast ,Mutation ,Schizosaccharomyces pombe ,biology.protein ,Schizosaccharomyces pombe Proteins ,Transcription Factors - Abstract
Mating-type switching in Schizosaccharomyces pombe entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, mat2-P or mat3-M, is chosen to convert the expressed mat1 locus. By measuring the biases in mat1 content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (mat2-M mat3-P) or when the SRE2 and SRE3 recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from mat2 and the SRE2 enhancer, towards mat3 and the SRE3 enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD+-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD+-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to mat2-P in M cells, perhaps by inhibiting the SRE3 recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.
- Published
- 2018
17. A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.
- Author
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Zdravković, Aleksandar, Daley, James M., Dutta, Arijit, Tatsuya Niwa, Yasuto Murayama, Shuji Kanamaru, Kentaro Ito, Takahisa Maki, Argunhan, Bilge, Masayuki Takahashi, Hideo Tsubouchi, Sung, Patrick, and Hiroshi Iwasaki
- Subjects
DOUBLE-strand DNA breaks ,DNA repair ,SCHIZOSACCHAROMYCES pombe ,PHOSPHORYLATION ,AMINO acids - Abstract
The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
18. CLASPs Are Required for Proper Microtubule Localization of End-Binding Proteins
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Irina Kaverina, Ashley D. Grimaldi, Dmitry Yampolsky, Benjamin P. Fitton, Ikuko Hayashi, Anne Straube, Michael W. Davidson, Tatyana Svitkina, Takahisa Maki, and Daniel Roth
- Subjects
Cytoplasm ,Microtubule-associated protein ,GTPase ,Plasma protein binding ,Biology ,Microtubules ,DNA-binding protein ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Tubulin ,Microtubule ,Animals ,Humans ,Molecular Biology ,Genetics ,Cell Biology ,Rats ,Cell biology ,Cell culture ,biology.protein ,Carrier Proteins ,Microtubule-Associated Proteins ,Protein Binding ,Developmental Biology - Abstract
SummaryMicrotubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that cytoplasmic linker-associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, whereas neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at preformed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells.
- Published
- 2014
- Full Text
- View/download PDF
19. CLASP2 Has Two Distinct TOG Domains That Contribute Differently to Microtubule Dynamics
- Author
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Ikuko Hayashi, Irina Kaverina, Ashley D. Grimaldi, Sotaro Fuchigami, and Takahisa Maki
- Subjects
Models, Molecular ,Molecular model ,Dimer ,Molecular Sequence Data ,macromolecular substances ,Crystallography, X-Ray ,Microtubules ,Protein Structure, Secondary ,Article ,Green fluorescent protein ,chemistry.chemical_compound ,Mice ,Structural Biology ,Microtubule ,Animals ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Gene ,Cells, Cultured ,Binding Sites ,biology ,Protein Structure, Tertiary ,Crystallography ,Tubulin ,HEK293 Cells ,chemistry ,biology.protein ,Biophysics ,Protein Multimerization ,Microtubule-Associated Proteins ,Function (biology) ,Protein Binding - Abstract
CLIP-associated proteins CLASPs are mammalian microtubule (MT) plus-end tracking proteins (+ TIPs) that promote MT rescue in vivo. Their plus-end localization is dependent on other + TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. MT association of CLASPs is suggested to be regulated by multiple TOG (tumor overexpressed gene) domains and by the serine-arginine (SR)-rich region, which contains binding sites for EB1. Here, we report the crystal structures of the two TOG domains of CLASP2. Both domains consist of six HEAT repeats, which are similar to the canonical paddle-like tubulin-binding TOG domains, but have arched conformations. The degrees and directions of curvature are different between the two TOG domains, implying that they have distinct roles in MT binding. Using biochemical, molecular modeling and cell biological analyses, we have investigated the interactions between the TOG domains and αβ-tubulin and found that each domain associates differently with αβ-tubulin. Our findings suggest that, by varying the degrees of domain curvature, the TOG domains may distinguish the structural conformation of the tubulin dimer, discriminate between different states of MT dynamic instability and thereby function differentially as stabilizers of MTs.
- Published
- 2014
20. A novel PAR-1-binding protein, MTCL1, plays critical roles in organizing microtubules in polarizing epithelial cells
- Author
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Yoshiko Amano, Hisashi Hirano, Masashi Akitsu, Akio Yamashita, Mariko Ide, Noriaki Arakawa, Kazunari Yamashita, Kyoko Shimada, Shigeo Ohno, Takahisa Maki, Yoshinori Sato, Ikuko Hayashi, and Atsushi Suzuki
- Subjects
Microtubule-associated protein ,Immunoprecipitation ,Plasma protein binding ,Protein Serine-Threonine Kinases ,Biology ,Transfection ,Microtubules ,Madin Darby Canine Kidney Cells ,Mice ,Dogs ,Microtubule ,Cell polarity ,Morphogenesis ,Animals ,Humans ,Cells, Cultured ,Epithelial polarity ,HEK 293 cells ,Cell Polarity ,Epithelial Cells ,Cell Biology ,Anatomy ,Cell biology ,HEK293 Cells ,Centrosome ,Microtubule-Associated Proteins ,HeLa Cells ,Protein Binding - Abstract
The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiled-coil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.
- Published
- 2013
21. Crystallization and preliminary X-ray data analysis of the pXO1 plasmid-partitioning factor TubZ from Bacillus cereus
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Shota Hoshino, Takahisa Maki, and Ikuko Hayashi
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GTP' ,Biophysics ,Bacillus cereus ,Biology ,Crystallography, X-Ray ,Biochemistry ,law.invention ,GTP Phosphohydrolases ,Plasmid ,Bacterial Proteins ,Structural Biology ,law ,Tubulin ,Genetics ,Crystallization ,FtsZ ,Plasmid partitioning ,Hydrolysis ,fungi ,Condensed Matter Physics ,biology.organism_classification ,Crystallography ,enzymes and coenzymes (carbohydrates) ,Crystallization Communications ,biological sciences ,biology.protein ,health occupations ,bacteria ,Guanosine Triphosphate ,Monoclinic crystal system ,Plasmids - Abstract
TubZ is a structural homologue of tubulin and FtsZ GTPases, which are involved in the type III plasmid-partitioning system. TubZ assembles into polymers in a GTP-dependent manner and drives plasmid segregation as ‘cytomotive’ filaments. In this study, C-terminally truncated TubZ from Bacillus cereus was crystallized in the presence or absence of GDP by the hanging-drop vapour-diffusion method. The crystal of TubZ in complex with GDP belonged to the monoclinic space group P21, with unit-cell parameters a = 67.05, b = 84.49, c = 67.66 A, β = 92.92°, and was non-isomorphous with GDP-bound TubZ previously crystallized in the presence of the slowly hydrolysable GTP analogue GTPγS. TubZ was also crystallized in the free form and the crystal belonged to space group P21, with unit-cell parameters a = 53.91, b = 65.54, c = 58.18 A, β = 106.19°. Data were collected to 1.7 and 2.1 A resolution for the free and GDP-bound forms, respectively.
- Published
- 2012
22. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules
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Chikashi Obuse, Takahisa Maki, Kanako Ozaki, Makoto Iimori, Toshiyuki Habu, Yasushi Hiraoka, Ikuko Hayashi, Tomohiro Matsumoto, and Yuji Chikashige
- Subjects
Models, Molecular ,Calponin ,Mutant ,Molecular Sequence Data ,Down-Regulation ,Aster (cell biology) ,Microtubules ,Serine ,Microtubule ,Schizosaccharomyces ,Amino Acid Sequence ,Threonine ,Phosphorylation ,biology ,Organisms, Genetically Modified ,Sequence Homology, Amino Acid ,Protein Stability ,Wild type ,Cell Biology ,Cell biology ,Biochemistry ,Mutation ,biology.protein ,Schizosaccharomyces pombe Proteins ,Microtubule-Associated Proteins ,Protein Kinases ,Protein Processing, Post-Translational - Abstract
Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.more » -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.« less
- Published
- 2011
23. The novel PAR-1-binding protein MTCL1 has crucial roles in organizing microtubules in polarizing epithelial cells.
- Author
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Yoshinori Sato, Masashi Akitsu, Yoshiko Amano, Kazunari Yamashita, Mariko Ide, Kyoko Shimada, Akio Yamashita, Hisashi Hirano, Noriaki Arakawa, Takahisa Maki, Ikuko Hayashi, Shigeo Ohno, and Atsushi Suzuki
- Subjects
CELL physiology ,MICROTUBULES ,EPITHELIAL cells ,N-terminal residues ,CENTROSOMES ,CROSSLINKING (Polymerization) - Abstract
The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiledcoil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
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