The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bioriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the ...intramitotic production of a diffusible inhibitor of APC/CCdc20 (the anaphase-promoting complex/cyclosome and its coactivator Cdc20, a large ubiquitin ligase). Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit “wait anaphase” signals that preserve genome integrity.
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•Mad1-Mad2 directs mitotic checkpoint complex (MCC) assembly during interphase•Interphase MCC assembly requires Mad1’s nuclear-pore-targeting domain and Tpr•Loss of interphase MCCs reduces mitotic timing, fidelity, and checkpoint robustness•Artificially tethering Mad1-Mad2 to nuclear pores restores normal M phase controls
A newly identified interphase checkpoint to prevent premature anaphase involves the tethering of checkpoint proteins Mad1-Mad2 to nuclear pores. This checkpoint is important for reinforcing the kinetochore-based checkpoint that kicks in after nuclear envelope breakdown as disabling it accelerates anaphase and increases chromosome segregation errors.
Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability and tumorigenesis. In ...response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi Anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and links timely genome replication to DNA repair.
The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex ...(MCC) 1, 2. During interphase, Mad1-Mad2 generates MCC at nuclear pores 3. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment 1, 2. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment 4–6. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial 7–12, and the specific phosphorylation event(s) that trigger corona formation remain elusive 5, 13. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ’s sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a “wait anaphase” signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.
•RZZ mediates a temporal switch in how Mad1-Mad2 is recruited to kinetochores•Mps1 phosphorylates Rod’s N terminus to trigger fibrous corona formation•Mad1-Mad2 requires a non-receptor activity of Bub1 to inhibit anaphase•Nonsense-associated alternative splicing can circumvent BUB1 disruption
Rodriguez-Rodriguez et al. identify distinct roles for Bub1, KNL1, and RZZ in SAC signaling and fibrous corona formation. They also show that BUB1-disrupted clones re-express Bub1 and regain SAC function via nonsense-associated alternative splicing, an often-overlooked transcriptional response that can limit penetrance in genome editing experiments
Chromothripsis: Chromosomes in Crisis Jones, Mathew J.K.; Jallepalli, Prasad V.
Developmental cell,
11/2012, Letnik:
23, Številka:
5
Journal Article
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During oncogenesis, cells acquire multiple genetic alterations that confer essential tumor-specific traits, including immortalization, escape from antimitogenic signaling, neovascularization, ...invasiveness, and metastatic potential. In most instances, these alterations are thought to arise incrementally over years, if not decades. However, recent progress in sequencing cancer genomes has begun to challenge this paradigm, because a radically different phenomenon, termed chromothripsis, has been suggested to cause complex intra- and interchromosomal rearrangements on short timescales. In this Review, we review established pathways crucial for genome integrity and discuss how their dysfunction could precipitate widespread chromosome breakage and rearrangement in the course of malignancy.
The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase ...Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1wt) or a mutant version (Mps1as) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20's association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B-dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.
Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage ...furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cohesin not only links sister chromatids but also inhibits the transcriptional machinery's interaction with and movement along chromatin. In contrast, replication forks must traverse such ...cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)-CTF18 clamp loader (RFCCTF18) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin's SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8-10) (including those derived from Roberts' syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin's hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFCCTF18. Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts' syndrome cohesinopathy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aneuploidy, an abnormal chromosome number, has been recognized as a hallmark of human cancer for nearly a century; however, the mechanisms responsible for this abnormality have remained elusive. Here ...we report the identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions. We show that genetic inactivation of hCDC4, by means of targeted disruption of the gene in karyotypically stable colorectal cancer cells, results in a striking phenotype associated with micronuclei and chromosomal instability. This phenotype can be traced to a defect in the execution of metaphase and subsequent transmission of chromosomes, and is dependent on cyclin E-a protein that is regulated by hCDC4 (refs 2-4). Our data suggest that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact ...human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.
Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint ...limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.
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•DDKi causes spontaneous fork stalling and inhibits late origin firing•ATRi and Chk1i rescue origin firing in DDKi cells and frog egg extracts•DDKi blocks RPA loading, nascent-strand protection, and restart at stalled forks•MERIT40 and PDS5B are key DDK substrates in nascent-strand protection and restart
Eukaryote genomes duplicate via spatially and temporally regulated firing of replication origins. Using chemical genetics and phosphoproteomics, Jones et al. show that human DDK promotes origin firing by counteracting the S phase checkpoint and enables the uncoupling, protection, and restart of stalled forks.
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