Biomedical subjects

Duncan J Clarke

Publications and source records attributed to Duncan J Clarke.

At least 19 recordsLinked to original sources

PubMed2006

PIASgamma is required for faithful chromosome segregation in human cells.

BACKGROUND: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. PRINCIPAL FINDINGS: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II. CONCLUSIONS: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.

Anaphase↗

PubMed2006

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae.

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.

Adenosine Triphosphatases↗

PubMed2006

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control.

BACKGROUND: Proteins containing ubiquitin-like (UBL) and ubiquitin associated (UBA) domains have been suggested to shuttle ubiquitinated substrates to the proteasome for degradation. There are three UBL-UBA containing proteins in budding yeast: Ddi1, Dsk2 and Rad23, which have been demonstrated to play regulatory roles in targeting ubiquitinated substrates to the proteasome for degradation. An involvement of these proteins in cell cycle related events has also been reported. We tested whether these three proteins act redundantly in the cell cycle. RESULTS: Here we show that the UBL-UBA proteins are partially redundant for cell cycle related roles. RAD23 is redundant with DDI1 and DSK2, but DDI1 and DSK2 are not redundant with each other and the triple deletion shows a synthetic effect, suggesting the existence of at least two roles for RAD23 in cell cycle control. The rad23Deltaddi1Deltadsk2Delta triple deletion strain delays both in G2/M-phase and in mid-anaphase at high temperatures with duplicated spindle pole bodies. Cell cycle progression in the triple deletion strain can only be partially rescued by a rad23 allele lacking the c-terminal UBA domain, suggesting that RAD23 requires its c-terminal UBA domain for full function. In addition to their ability to bind ubiquitin and the proteasome, the UBL-UBA proteins also share the ability to homodimerize. Rad23 and Dsk2 dimerization requires their UBL and/or UBA domains whereas Ddi1 dimerization does not. Here we show that Ddi1 homodimerization is necessary for its cell cycle related functions. CONCLUSION: The three yeast UBL-UBA proteins have partially redundant roles required for progression through mitosis.

Journal Article↗

PubMed2006

Topoisomerase II checkpoints: universal mechanisms that regulate mitosis.

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.

Chromosomes, Human↗

PubMed2006

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast.

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Cell Cycle Proteins↗

PubMed2006

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin.

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain.

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.

Animals↗

PubMed2005

Anaphase promoting complex or cyclosome?

The anaphase promoting complex/cyclosome (APC/C) was initially described as a multi-subunit protein complex that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes to initiate loss of sister chromatid cohesion. However, recent studies have identified important new functions of the APC/C. Moreover, sister centromere separation can occur in the absence of APC/C activity in mammals, indicating that anaphase onset might be triggered by multiple factors. Here we discuss whether the APC/C functions primarily as the anaphase trigger, or whether it has more general properties, relevant for cell cycle control at multiple developmental and cell cycle stages. Additionally, we discuss the validity of the APC-dependent model for sister segregation in mammals.

Anaphase↗

PubMed2005

Proteasome activity is required for centromere separation independently of securin degradation in human cells.

Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear. Therefore, we asked if proteasome activity is needed for anaphase onset in human cells that lack securin. We find that even in the absence of securin, a metaphase block with cohered sister centromeres can be enforced in the absence of proteasome activity. Therefore, other targets of the proteasome must be degraded to allow anaphase onset.

Anaphase↗

PubMed2005

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.

Anaphase↗

PubMed2005

Separase is required at multiple pre-anaphase cell cycle stages in human cells.

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2-M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.

Cell Cycle↗

PubMed2005

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

MRX (Mre11/Rad50/Xrs2) mutants reveal dual intra-S-phase checkpoint systems in budding yeast.

The intra-S-phase checkpoint is a signaling pathway that induces slow DNA replication in the presence of DNA damage. In humans, defects in this checkpoint pathway might account for phenotypes seen in autosomal recessive diseases including ataxia telangiectasia-like disorder and Nijmegen breakage syndrome, where MRN complex components,Mre11 and Nbs1, are mutated. Here we provide evidence that the equivalent budding yeast complex, MRX (Mre11/Rad50/Xrs2), is not required for the intra-S-phase checkpoint in response to DNA alkylation damage, but is required in the presence of double-stranded DNA breaks. These data indicate, at least in budding yeast, that alternate pathways enforce replication slowing depending on the particular DNA lesion.

Bleomycin↗

PubMed2004

p53 prevents the accumulation of double-strand DNA breaks at stalled-replication forks induced by UV in human cells.

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in nucleotide-excision-repair (NER)-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase epsilon, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol eta. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV, all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.

Cell Line, Tumor↗

PubMed2004

Cdc20 in S-phase: the Banquo at replication's banquet.

The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that covalently attaches ubiquitins onto proteins to target them for proteolysis by the 26S proteasome. During mitosis, the APC/C is instrumental in allowing the cell to enter and exit from mitosis. The APC/C accomplishes this by using different specificity factors to recognize, interact with, and ubiquitylate key proteins that block cell cycle progression. The specificity factors, Cdc20p and Cdh1p, are not always associated with the APC/C and indeed they have the ability to interact with substrates in isolation. The molecular events that take place in order for Cdc20p and Cdh1p to couple substrates and APC/C are currently being resolved. Meanwhile, evidence has emerged suggesting that at least one of the specificity factors, Cdc20p, might be capable of functioning independently of the APC/C.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

S-phase checkpoint controls mitosis via an APC-independent Cdc20p function.

Cells divide with remarkable fidelity, allowing complex organisms to develop and possess longevity. Checkpoint controls contribute by ensuring that genome duplication and segregation occur without error so that genomic instability, associated with developmental abnormalities and a hallmark of most human cancers, is avoided. S-phase checkpoints prevent cell division while DNA is replicating. Budding yeast Mec1p and Rad53p, homologues of human checkpoint kinases ATM/ATR and Chk2, are needed for this control system. How Mec1p and Rad53p prevent mitosis in S phase is not known. Here we provide evidence that budding yeasts avoid mitosis during S phase by regulating the anaphase-promoting complex (APC) specificity factor Cdc20p: Mec1p and Rad53p repress the accumulation of Cdc20p in S phase. Because precocious Cdc20p accumulation causes anaphase onset and aneuploidy, Cdc20p concentrations must be precisely regulated during each and every cell cycle. Catastrophic mitosis induced by Cdc20p in S phase occurs even in the absence of core APC components. Thus, Cdc20p can function independently of the APC.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Establishment of dependence relationships between genome replication and mitosis.

Although budding yeast cell biology and genetics provided a powerful system to isolate S-phase checkpoint mutants, initial studies relied on a defect not likely to be relevant in higher eukaryotes. The first mutants were isolated for their inability to restrain mitotic spindle elongation in S-phase. Since most eukaryotes do not assemble spindles until prometaphase the validity of this approach might have been questioned. However, these early studies were designed with a highly valid assumption in mind; that checkpoints have a variety of targets, but comprise conserved kinase cascades that make up these signaling pathways. The task that lies ahead is to determine targets of the S-phase checkpoint relevant to mammals. One step forward might be the realization that the budding yeast S-phase checkpoint prevents loss of sister chromatid cohesion while DNA replication is ongoing. If this mechanism is conserved in mammals, it could prove vital for chromosome segregation fidelity.

Animals↗

PubMed2003

p53-independent checkpoint controls in a plant cell model.

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpoint-adapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.

Cell Cycle↗