Checkpoint adaptation was originally defined in yeast as the ability to divide despite the presence of damaged DNA. An important unanswered question is whether checkpoint adaptation also occurs in human cells. Here, we show that following the ionizing radiation-induced G 2 checkpoint, human osteosarcoma cells entered mitosis with ;-H2AX foci, a marker for unrepaired DNA double-strand breaks. Exit from the G 2 checkpoint was accelerated by inhibiting the checkpoint kinase 1 (Chk1) and delayed by overexpressing wild-type Chk1 or depleting the Polo-like kinase 1 (Plk1). Chk1 and Plk1 controlled this process, at least partly, via independent signaling pathways. Our results suggest that human cells are able to exit the checkpoint arrest and divide before the damage has been fully repaired. Such cell division in the presence of damaged DNA may be detrimental for genetic stability and could potentially contribute to cancer development. (Cancer Res 2006; 66(21): 10253-7)

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.When DNA is damaged or chromosomes are incompletely replicated, cells become checkpoint arrested. These checkpoints avoid replication of damaged template DNA and prevent aberrant segregation of damaged or partly replicated chromosomes. In budding yeast, proteolysis of anaphase inhibitors is regulated by these checkpoint systems. Progression from metaphase to anaphase is inhibited by Pds1p in Saccharomyces cerevisiae (6,7,29,30). Before anaphase, Pds1p binds to Esp1p, inhibiting its anaphase-promoting activity (3). During an unperturbed cell cycle, Pds1p becomes polyubiquitinated at the metaphase-to-anaphase transition by multienzyme anaphase-promoting complex (APC)-cyclosome complexes. The modified forms are then recognized and degraded by 26S proteasomes (7). Once released from Pds1p, Esp1p activity induces the onset of anaphase.pds1 mutants fail to execute checkpoint control in response to DNA damage, spindle poisons, or replication inhibition (4, 29, 30). Pds1p is required for replication checkpoint control only late in S phase, not in the context of an early S-phase replication block enforced by hydroxyurea (HU) (4,29,30). In the presence of 0.1 M HU, replication proceeds more slowly. Under these conditions, cells perform other aspects of cell cycle progression, budding, and spindle assembly as rapidly as in the absenc...

Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.

The functional domain structure of human DNA topoisomerase II␣ and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase II␣ and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase II␣ is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme involved in regulating the conversion of DNA between different topological isoforms (42, 60). It fulfills essential functions in DNA replication (41, 53) and chromosome segregation during both mitosis (26, 27) and meiosis (46), and it is thought to play a key role in certain types of DNA recombination events (8,10,22,30,48). The enzyme has furthermore been suggested to constitute a component of nuclear scaffold structures (3, 23), where it may be involved in chromosome condensation (4, 58) and decondensation (45).Topoisomerase II binds to DNA as a dimer and cleaves its DNA substrate with a 4-bp stagger (40, 60) at sites with loosely defined sequences (54). Upon DNA cleavage, each subunit of topoisomerase II becomes transiently ligated to the 5Ј ends of the respective DNA strands through an O 4 -phosphotyrosine bond involving a tyrosine residue in the active site of the protein (7, 36). Catalytic activity is ATP dependent, and it is performed by passing one intact DNA helix through the enzyme-mediated double-stranded DNA break, followed by rejoining of the DNA strands (42).Although the mechanics of topoisomerase II function have been characterized in great detail, little information has emerged about the functional organization of the enzyme. Proteolysis studies on purified topoisomerase II from Saccharomyces cerevisiae (35), Schizosaccharomyces pombe (50), Drosophila melanogaster (34), and humans (9) suggest that the enzyme is composed of three major structural domains, of which two are constituted by the conserved N-terminal and central parts of the protein and a third is constituted by the highly divergent C-terminal region.The fragment originating from the N-...

In budding yeast, activation of the small Ras-like GTPase Tem1 triggers exit from mitosis and cytokinesis. Tem1 is regulated by Bub2/Bfa1, a two-component GTPase-activating protein (GAP), and by Lte1, a putative guanine nucleotide exchange factor. Lte1 is confined to the bud cortex, and its spatial separation from Tem1 at the spindle pole body (SPB) is important to prevent untimely exit from mitosis. The pathways contributing to Lte1 asymmetry have not been elucidated. Here we show that establishment of Lte1 at the cortex occurs by an actin-independent mechanism, which requires activation of Cdc28/Cln kinase at START and Cdc42, a key regulator of cell polarity and cytoskeletal organisation. This defines a novel role for Cdc42 in late mitotic events. In turn, dissociation of Lte1 from the cortex in telophase depends on activation of the Cdc14 phosphatase. Ectopic expression of Cdc14 at metaphase results in premature dephosphorylation of Lte1 coincident with its release from the cortex. In vitro phosphatase assays confirm that Lte1 is a direct substrate for Cdc14. Our results suggest that the asymmetry in Lte1 localisation is imposed by Cdc28-dependent phosphorylation.Finally, we report a mutational analysis undertaken to investigate intrinsic Lte1 determinants for localisation. Our data suggest that an intrameric interaction between the N-and C-terminal regions of Lte1 is important for cortex association.

No abstract

In Saccharomyces cerevisiae, the metaphase–anaphase transition is initiated by the anaphase-promoting complex–dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase–anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Genetic evidence suggests that the securin Pds1p is the target of a late-S-phase checkpoint control. Here we show that Pds1p becomes essential once two-thirds of the genome has been replicated and that the coupling of the completion of genome replication with mitosis relies on the regulation of Pds1p levels. Mec1p is needed to maintain Pds1p levels under S-phase checkpoint conditions. In contrast, Rad53p and Chk1p, needed for the stabilization of Pds1p in the context of the G2 DNA-damage checkpoint pathway, are dispensable. Thus, the Pds1p-dependent late-S-phase checkpoint pathway couples replication with mitosis but is mechanistically distinct from the G2 DNA-damage checkpoint. Finally, we show that the inhibition of spindle elongation in early S phase, controlled by the Mec1p/Rad53p branch, is not regulated via Pds1p/Esp1p. This can mechanistically explain the need for branched S-phase checkpoint controls.