The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Robert V. Skibbens - One of the best experts on this subject based on the ideXlab platform.
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the spindle pole body assembly component mps3p nep98p functions in sister chromatid cohesion
Journal of Biological Chemistry, 2004Co-Authors: Lisa M. Antoniacci, Peter Uetz, Stanley Fields, Margaret A. Kenna, Robert V. SkibbensAbstract:Abstract For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister Chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister Chromatids are paired together, identifying the products of replication as sister Chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.
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The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion.
Journal of Biological Chemistry, 2004Co-Authors: Lisa M. Antoniacci, Peter Uetz, Stanley Fields, Margaret A. Kenna, Robert V. SkibbensAbstract:For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister Chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister Chromatids are paired together, identifying the products of replication as sister Chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.
Mitsuhiro Yanagida - One of the best experts on this subject based on the ideXlab platform.
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Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast.
Current biology : CB, 2002Co-Authors: Yusuke Toyoda, Koji Nagao, Kanji Furuya, Gohta Goshima, Kohta Takahashi, Mitsuhiro YanagidaAbstract:Proteins conserved from yeast to human hold two sister Chromatids together. The failure to form cohesion in the S phase results in premature separation of Chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.
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requirement of chromatid cohesion proteins rad21 scc1 and mis4 scc2 for normal spindle kinetochore interaction in fission yeast
Current Biology, 2002Co-Authors: Yusuke Toyoda, Koji Nagao, Kanji Furuya, Gohta Goshima, Kohta Takahashi, Mitsuhiro YanagidaAbstract:Abstract Background: Proteins conserved from yeast to human hold two sister Chromatids together. The failure to form cohesion in the S phase results in premature separation of Chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. Results: The loss of chromatid cohesion in fission yeast mutants ( mis4-242 and rad21-K1 ) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17 , which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. Conclusions: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.
Lisa M. Antoniacci - One of the best experts on this subject based on the ideXlab platform.
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the spindle pole body assembly component mps3p nep98p functions in sister chromatid cohesion
Journal of Biological Chemistry, 2004Co-Authors: Lisa M. Antoniacci, Peter Uetz, Stanley Fields, Margaret A. Kenna, Robert V. SkibbensAbstract:Abstract For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister Chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister Chromatids are paired together, identifying the products of replication as sister Chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.
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The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion.
Journal of Biological Chemistry, 2004Co-Authors: Lisa M. Antoniacci, Peter Uetz, Stanley Fields, Margaret A. Kenna, Robert V. SkibbensAbstract:For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister Chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister Chromatids are paired together, identifying the products of replication as sister Chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.
Becky Alhadeff - One of the best experts on this subject based on the ideXlab platform.
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Analysis of sister-chromatid exchanges.
Current protocols in human genetics, 2001Co-Authors: J German, Becky AlhadeffAbstract:Two requirements for the cytogenetic analysis of sister-chromatid exchanges (SCEs) in somatic cells are (1) a population of actively proliferating cells that will provide an adequate number of metaphases and (2) sister Chromatids that in some way are differentially labeled or stained in the metaphases. SCEs can be recognized as abrupt discontinuities in the staining patterns of the two Chromatids of a metaphase chromosome at what appear to be identical sites, with reciprocal switching from one chromatid to its sister. This protocol uses phytohemagglutinin (PHA)-stimulated cultures of blood lymphocytes as a source of proliferating cells. The cells are incubated with the thymidine analog BrdU. Slides prepared from fixed cells with BrdU-substituted chromosomes are treated with Hoechst 33258, exposed to light and heat, and then Giemsa-stained to produce differentially stained chromosomes. The Chromatids with bifilar substitution exhibit a lighter purple stain than their unifilarly substituted sister Chromatids.
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Current Protocols in Human Genetics - Analysis of sister-chromatid exchanges
Current Protocols in Human Genetics, 1994Co-Authors: James German, Becky AlhadeffAbstract:Two requirements for the cytogenetic analysis of sister-chromatid exchanges (SCEs) in somatic cells are (1) a population of actively proliferating cells that will provide an adequate number of metaphases and (2) sister Chromatids that in some way are differentially labeled or stained in the metaphases. SCEs can be recognized as abrupt discontinuities in the staining patterns of the two Chromatids of a metaphase chromosome at what appear to be identical sites, with reciprocal switching from one chromatid to its sister. This protocol uses phytohemagglutinin (PHA)-stimulated cultures of blood lymphocytes as a source of proliferating cells. The cells are incubated with the thymidine analog BrdU. Slides prepared from fixed cells with BrdU-substituted chromosomes are treated with Hoechst 33258, exposed to light and heat, and then Giemsa-stained to produce differentially stained chromosomes. The Chromatids with bifilar substitution exhibit a lighter purple stain than their unifilarly substituted sister Chromatids.
Job Dekker - One of the best experts on this subject based on the ideXlab platform.
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detecting chromatin interactions between and along sister Chromatids with sisterc
Nature Methods, 2020Co-Authors: Marlies E Oomen, Adam K Hedger, Jonathan K Watts, Job DekkerAbstract:Chromosome segregation requires both compaction and disentanglement of sister Chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister Chromatids. SisterC employs 5-bromo-2′-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister Chromatids at centromeres. Along arms, sister Chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister Chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis. It remains impossible using conventional Hi-C to differentiate interactions between and along sister Chromatids. SisterC relies on selective destruction of nascent DNA and in combination with Hi-C offers a means to study intra- and inter-sister interactions independently.
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detecting chromatin interactions along and between sister Chromatids with sisterc preprint
bioRxiv, 2020Co-Authors: Marlies E Oomen, Adam K Hedger, Jonathan K Watts, Job DekkerAbstract:Accurate chromosome segregation requires chromosome compaction with concordant disentanglement of the two sister Chromatids. This process has been studied extensively by microscopy but has remained a challenge for genomic methods, such as Hi-C, because sister Chromatids have identical DNA sequences. Here we describe SisterC, a chromosome conformation capture assay that can distinguish interactions between and within sister Chromatids. The assay is based on BrdU incorporation during S-phase, which labels the newly replicated strands of the sister Chromatids. This is followed by Hi-C, e.g. during different stages of mitosis, and the selective destruction of BrdU containing strands by UV/Hoechst treatment. After PCR amplification and sequencing of the remaining intact strands, this allows for the assignment of Hi-C products as inter- and intra-sister interactions by read orientation. We performed SisterC on mitotically arrested S. cerevisiae cells. As expected, we find prominent interactions and alignment of sister Chromatids at their centromeres. Along the arms, sister Chromatids are less precisely aligned with inter-sister connections every ~35kb. In many instances, inter-sister interactions do not involve the interaction of two identical loci but occur between cohesin binding sites that can be offset by 5 to 25kb. Along sister Chromatids, extruding cohesin forms loops up to 50kb. Combined, SisterC allows the observation of the complex interplay between sister chromatid compaction and sister chromatid segregation as the cell transitions from late S-phase to mitosis. SisterC should be applicable to study mitotic events in a wide range of organisms and cell types.