PATTERN OF SEGREGATION OF LABELED DNA AT THE CHROMATID LEVEL OF CHROMOSOMES WITH DIFFUSE CENTROMERES

1969 ◽  
Vol 11 (4) ◽  
pp. 928-936 ◽  
Author(s):  
Janet S. R. Nelson
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sister Chromatid ◽  
Cycle Time ◽  
Full Length ◽  
Sister Chromatids ◽  
Labeling Pattern ◽  
Shoot Meristems

After pulse labeling Luzula purpurea shoot meristems with3H-thymidine, the cell cycle and its subdivisions were determined autoradiographically using the labeled mitoses method. The cycle time was 20 hr. G1 = 4.3 hr; C = 9.5 hr; G2 = 2.9 hr; and M = 3.3 hr. In subsequent experiments seedlings were labeled for 4 to 5 hr, long enough to label the chromosomes' full length, eliminating problems associated with asynchronous DNA replication. The longer labeling period did not alter the cell cycle parameters.Cells were designated as being in the first or second division after labeling by the time from the midpoint of the labeling period. Seedlings were placed in 0.1% cycloheximide solution to cause sister chromatids in metaphase cells to separate from each other. Autoradiographs were then made to determine segregation of labeled DNA at the chromatid level. At the first division after labeling all chromatids appeared to be labeled. At the second division after labeling with 3H-thymidine the labeling pattern was consistent with semiconservative segregation of labeled DNA and with sister chromatid exchange.These results are discussed in relation to cytological and photometric studies on chromosome strandedness in Luzula and in other organisms with chromosomes with diffuse centromeres.

scholarly journals Dynamics of sister chromatids through the cell cycle: Together and apart

2018 ◽  
Vol 217 (6) ◽  
pp. 1887-1889 ◽  
Author(s):  
Motoko Takahashi ◽  
Toru Hirota
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sister Chromatid ◽  
Fundamental Question ◽  
Live Cells ◽  
Sister Chromatids ◽  
Cell Biol

When and how sister chromatid resolution occurs after DNA replication is a fundamental question. Stanyte et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801157) used CRISPR/Cas9 technology to label and track genomic loci in live cells throughout the cell cycle, shedding light on how replication is linked to mitotic sister chromatid organization.

scholarly journals Dynamics of sister chromatid resolution during cell cycle progression

2018 ◽  
Author(s):  
Rugile Stanyte ◽  
Johannes Nuebler ◽  
Claudia Blaukopf ◽  
Rudolf Hoefler ◽  
Roman Stocsits ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sister Chromatid ◽  
Cell Cycle Progression ◽  
Human Cells ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Dividing Cells ◽  
G2 Phase ◽  
Almost All

Faithful genome transmission in dividing cells requires that the two copies of each chromosome’s DNA package into separate, but physically linked, sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. Here, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labelling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase, irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication, and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Further, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.

scholarly journals Dynamics of sister chromatid resolution during cell cycle progression

2018 ◽  
Vol 217 (6) ◽  
pp. 1985-2004 ◽  
Author(s):  
Rugile Stanyte ◽  
Johannes Nuebler ◽  
Claudia Blaukopf ◽  
Rudolf Hoefler ◽  
Roman Stocsits ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sister Chromatid ◽  
Cell Cycle Progression ◽  
Human Cells ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Dividing Cells ◽  
G2 Phase ◽  
Almost All

Faithful genome transmission in dividing cells requires that the two copies of each chromosome’s DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Furthermore, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.

scholarly journals Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast

2001 ◽  
Vol 155 (5) ◽  
pp. 711-718 ◽  
Author(s):  
Fedor Severin ◽  
Anthony A. Hyman ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Budding Yeast ◽  
Sister Chromatid Cohesion ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Spindle Elongation

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

DETERMINATION OF CELL CYCLE IN PLANTS USING BrdU-FPG

1980 ◽  
Vol 22 (2) ◽  
pp. 305-308 ◽  
Author(s):  
Kirby J. Evans ◽  
W. Gary Filion
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cycle Time ◽  
Differential Staining ◽  
Root Tip ◽  
Cell Cycle Time ◽  
Sister Chromatids ◽  
Vicia Faba L ◽  
Established Cell

The BrdU-FPG method for differential staining of sister chromatids was employed to estimate the cell cycle time in two plants species. The protocol is based upon the ability to differentiate cytologically between chromosomes which have completed two or three DNA synthesis phases. Vicia faba L. which has a well established cell cycle time was used to verify the technique. Utilizing this method the cell cycle time of Zebrina pendula Schnizl. root tip meristem cells was determined to be 17.0 hours.

scholarly journals Multivalent interaction of ESCO2 with the replication machinery is required for sister chromatid cohesion in vertebrates

2019 ◽  
Vol 117 (2) ◽  
pp. 1081-1089 ◽  
Author(s):  
Dawn Bender ◽  
Eulália Maria Lima Da Silva ◽  
Jingrong Chen ◽  
Annelise Poss ◽  
Lauren Gawey ◽  
...  
Keyword(s):  
Dna Replication ◽  
Sister Chromatid ◽  
Sister Chromatid Cohesion ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
N Terminus ◽  
Mcm Complex ◽  
Unique Ability

The tethering together of sister chromatids by the cohesin complex ensures their accurate alignment and segregation during cell division. In vertebrates, sister chromatid cohesion requires the activity of the ESCO2 acetyltransferase, which modifies the Smc3 subunit of cohesin. It was shown recently that ESCO2 promotes cohesion through interaction with the MCM replicative helicase. However, ESCO2 does not significantly colocalize with the MCM complex, suggesting there are additional interactions important for ESCO2 function. Here we show that ESCO2 is recruited to replication factories, sites of DNA replication, through interaction with PCNA. We show that ESCO2 contains multiple PCNA-interaction motifs in its N terminus, each of which is essential to its ability to establish cohesion. We propose that multiple PCNA-interaction motifs embedded in a largely flexible and disordered region of the protein underlie the unique ability of ESCO2 to establish cohesion between sister chromatids precisely as they are born during DNA replication.

scholarly journals Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP

2003 ◽  
Vol 77 (22) ◽  
pp. 11992-12001 ◽  
Author(s):  
Zhong Deng ◽  
Constandache Atanasiu ◽  
John S. Burg ◽  
Dominique Broccoli ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Dominant Negative ◽  
Full Length ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr ◽  
Dyad Symmetry

ABSTRACT Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5- to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G1/S while TRF1 binding peaks in G2/M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.

scholarly journals Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity

2007 ◽  
Vol 178 (3) ◽  
pp. 345-354 ◽  
Author(s):  
Kerstin H. Thein ◽  
Julia Kleylein-Sohn ◽  
Erich A. Nigg ◽  
Ulrike Gruneberg
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Cysteine Protease ◽  
Mitotic Spindle ◽  
Regulatory Network ◽  
Successful Completion ◽  
Sister Chromatids ◽  
Multipolar Spindles ◽  
Chromatid Cohesion

Faithful chromosome segregation in mitosis requires the formation of a bipolar mitotic spindle with stably attached chromosomes. Once all of the chromosomes are aligned, the connection between the sister chromatids is severed by the cysteine protease separase. Separase also promotes centriole disengagement at the end of mitosis. Temporal coordination of these two activities with the rest of the cell cycle is required for the successful completion of mitosis. In this study, we report that depletion of the microtubule and kinetochore protein astrin results in checkpoint-arrested cells with multipolar spindles and separated sister chromatids, which is consistent with untimely separase activation. Supporting this idea, astrin-depleted cells contain active separase, and separase depletion suppresses the premature sister chromatid separation and centriole disengagement in these cells. We suggest that astrin contributes to the regulatory network that controls separase activity.

scholarly journals The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3455
Author(s):  
Janne J.M. van Schie ◽  
Job de Lange
Keyword(s):  
Dna Replication ◽  
Sister Chromatid ◽  
Molecular Mechanisms ◽  
Replication Stress ◽  
Sister Chromatid Cohesion ◽  
Key Factors ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Dna Replication Stress ◽  
Subsequent Stabilization

The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

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