scholarly journals Condensin facilitates sister chromatid separation in actively transcribed DNA regions by relieving the obstructive effect of transcription

2019 ◽  
Author(s):  
Norihiko Nakazawa ◽  
Orie Arakawa ◽  
Mitsuhiro Yanagida
Keyword(s):  
Sister Chromatid ◽  
Gene Locus ◽  
Time Lapse ◽  
Chromosomal Dna ◽  
Ssdna Binding ◽  
Genome Wide ◽  
Sister Chromatid Separation ◽  
Mitotic Chromosome Condensation ◽  
Ssdna Binding Proteins

AbstractThe evolutionarily conserved protein complex, condensin, is central to chromosome dynamics, including mitotic chromosome condensation and segregation. Genome-wide localization of condensin is correlated with transcriptional activity; however, the significance of condensin accumulation in transcribed regions remains unclear. Here, we demonstrate that condensin relieves the obstructive effect of mitotic transcription on sister chromatid separation in fission yeast, Schizosaccharomyces pombe. Time-lapse visualization of sister chromatid DNA separation revealed that mutant condensin causes delayed segregation specifically at mitotically transcribed, condensin-bound gene locus, ecm33+. Contrarily, the delay was abolished by transcriptional shut-off of the actively transcribed gene. We also showed that delayed separation at a heat shock-inducible gene locus, ssa1+, in condensin mutants was significantly alleviated by deletion of the gene. Since condensin has ability to remove ssDNA-binding proteins and RNA from unwound ssDNAs or DNA-RNA hybrids in vitro, we propose a model that condensin-mediated removal of mitotic transcripts from chromosomal DNA is the primary mechanism of sister chromatid separation.

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Author(s):  
Hongshan Zhang ◽  
Jeffrey M. Schaub ◽  
Ilya J. Finkelstein
Keyword(s):  
Protein A ◽  
Negative Regulator ◽  
Cellular Biology ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

AbstractRADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) activities is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using singlemolecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

scholarly journals Condensin locates at transcriptional termination sites in mitosis, possibly releasing mitotic transcripts

Open Biology ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 190125 ◽  
Author(s):  
Norihiko Nakazawa ◽  
Orie Arakawa ◽  
Mitsuhiro Yanagida
Keyword(s):  
Transcriptional Activation ◽  
Temperature Sensitive ◽  
Growth Defects ◽  
Transcriptional Termination ◽  
Genome Wide ◽  
Transcription Sites ◽  
Cold Sensitive ◽  
Inducible Genes ◽  
Mitotic Chromosome Condensation

Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast Schizosaccharomyces pombe . Combining the auxin-degron strain with the nda3 β-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5′-3′ exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3′-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3′-end may be a prerequisite for faithful chromosome segregation.

scholarly journals Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

1992 ◽  
Vol 117 (5) ◽  
pp. 921-934 ◽  
Author(s):  
CE Shamu ◽  
AW Murray
Keyword(s):  
Sister Chromatid ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Egg Extracts ◽  
Chromosome Separation ◽  
Sister Chromatid Separation ◽  
Sperm Nuclei ◽  
Cytostatic Factor

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Vol 48 (14) ◽  
pp. 7834-7843
Author(s):  
Hongshan Zhang ◽  
Jeffrey M Schaub ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Negative Regulator ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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