scholarly journals Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells

2009 ◽  
Vol 187 (2) ◽  
pp. 165-173 ◽  
Author(s):  
Silvia Canudas ◽  
Susan Smith
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromosome Morphology ◽  
Differential Regulation ◽  
S Phase ◽  
Human Cells ◽  
Genomic Integrity ◽  
Sister Chromatids ◽  
And Function ◽  
Telomeric Protein

Replicated sister chromatids are held together until mitosis by cohesin, a conserved multisubunit complex comprised of Smc1, Smc3, Scc1, and Scc3, which in vertebrate cells exists as two closely related homologues (SA1 and SA2). Here, we show that cohesinSA1 and cohesinSA2 are differentially required for telomere and centromere cohesion, respectively. Cells deficient in SA1 are unable to establish or maintain cohesion between sister telomeres after DNA replication in S phase. The same phenotype is observed upon depletion of the telomeric protein TIN2. In contrast, in SA2-depleted cells telomere cohesion is normal, but centromere cohesion is prematurely lost. We demonstrate that loss of telomere cohesion has dramatic consequences on chromosome morphology and function. In the absence of sister telomere cohesion, cells are unable to repair chromatid breaks and suffer sister telomere loss. Our studies elucidate the functional distinction between the Scc3 homologues in human cells and further reveal an essential role for sister telomere cohesion in genomic integrity.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

2011 ◽  
Vol 193 (6) ◽  
pp. 995-1007 ◽  
Author(s):  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Deoxyribonucleic Acid ◽  
S Phase ◽  
Human Cells ◽  
Target Sequence ◽  
Cyclin Dependent Kinase ◽  
Functional Importance ◽  
Interacting Protein ◽  
Egg Extracts

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

scholarly journals Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

2018 ◽  
Author(s):  
Alan Gao ◽  
Nicolai B. Larsen ◽  
Justin L. Sparks ◽  
Irene Gallina ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Lesions ◽  
List Type ◽  
Genomic Integrity ◽  
Dna Metabolism ◽  
Single Stranded Dna ◽  
Egg Extracts ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

SummaryDNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.HighlightsThe proteasome, in addition to SPRTN, degrades DPCs during DNA replicationProteasome-dependent DPC degradation requires DPC ubiquitylationDPC ubiquitylation is triggered by ssDNA and does not require the replisomeSPRTN-dependent DPC degradation is a post-replicative process

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 SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

2020 ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

scholarly journals Condensin in Chromatid Cohesion and Segregation

2015 ◽  
Vol 147 (4) ◽  
pp. 212-216 ◽  
Author(s):  
Susumu Uchiyama ◽  
Kiichi Fukui
Keyword(s):  
Protein Complex ◽  
Genomic Dna ◽  
Centromeric Region ◽  
Chromosome Morphology ◽  
S Phase ◽  
Phase 2 ◽  
Major Function ◽  
Sister Chromatids ◽  
Chromatid Cohesion

After replication of genomic DNA during the S phase, 2 chromatids hold together longitudinally. When cells enter mitosis, the paired sister chromatids start to condense and then segregate into individual chromatids except for the centromeric region. Upon attachment of microtubules to the kinetochore, subsequent pulling of the 2 sister chromatids by the spindles towards opposite poles results in 2 completely separated chromatids. Besides more than 100 kinds of kinetochore proteins, several key proteins such as cohesin, separase, shugoshin, and condensin contribute to chromatid cohesion and segregation. Among these proteins, condensin, a protein complex composed of 5 subunits discovered 2 decades ago, has been extensively studied in terms of the maintenance of chromosome morphology as its major function. Recent studies on condensin uncovered its role in chromatid cohesion and segregation, which will be reviewed in this article.

scholarly journals The Cdc6 Nucleotide–Binding Site Regulates Its Activity in DNA Replication in Human Cells

1999 ◽  
Vol 10 (8) ◽  
pp. 2631-2645 ◽  
Author(s):  
Utz Herbig ◽  
Clinton A. Marlar ◽  
Ellen Fanning
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Essential Role ◽  
Active Form ◽  
Human Cells ◽  
Amino Acid Substitutions ◽  
Human Homologue ◽  
Mutant Proteins ◽  
Initiation Of Dna Replication ◽  
Atp Binding And Hydrolysis

The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.

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