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To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH’s functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.

Ultrafine anaphase bridges (UFBs) are a potential source of genome instability that is a hallmark of cancer. UFBs can arise from DNA catenanes at centromeres/rDNA loci, late replication intermediates induced by replication stress, and DNA linkages at telomeres. Recently, it was reported that DNA intertwinements generated by homologous recombination give rise to a new class of UFBs, which have been termed homologous recombination ultrafine bridges (HR-UFBs). HR-UFBs are decorated with PICH and BLM in anaphase, and are subsequently converted to RPA-coated, single-stranded DNA bridges. Breakage of these sister chromatid entanglements leads to DNA damage that can be repaired by non-homologous end joining in the next cell cycle, but the potential consequences include DNA rearrangements, chromosome translocations and fusions. Visualisation of these HR-UFBs, and knowledge of how they arise, provides a molecular basis to explain how upregulation of homologous recombination or failure to resolve recombination intermediates leads to the development of chromosomal instability observed in certain cancers.

Successful chromosome segregation depends on the timely removal of DNA recombination and replication intermediates that interlink sister chromatids. These intermediates are acted upon by structure-selective endonucleases that promote incisions close to the junction point. GEN1, a member of the Rad2/XPG endonuclease family, was identified on the basis of its ability to cleave Holliday junction recombination intermediates. Resolution occurs by a nick and counter-nick mechanism in strands that are symmetrically related across the junction point, leading to the formation of ligatable nicked duplex products. The actions of GEN1 are, however, not restricted to HJs, as 5'-flaps and replication fork structures also serve as excellent in vitro substrates for the nuclease. In the cellular context, GEN1 activity is observed late in the cell cycle, as most of the protein is excluded from the nucleus, such that it gains access to DNA intermediates after the breakdown of nuclear envelope. Nuclear exclusion ensures the protection of replication forks and other DNA secondary structures important for normal metabolic processes. In this chapter, we describe the purification of recombinant GEN1 and detail biochemical assays involving the use of synthetic DNA substrates and cruciform-containing plasmids.

The efficient processing of homologous recombination (HR) intermediates, which often contain four-way structures known as Holliday junctions (HJs), is required for proper chromosome segregation at mitosis. Eukaryotic cells possess three distinct pathways of resolution: (i) HJ dissolution mediated by BLM-topoisomerase IIIα-RMI1-RMI2 (BTR) complex, and HJ resolution catalyzed by either (ii) SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX complex) or (iii) GEN1. The BTR pathway acts at all times throughout the cell cycle, whereas the actions of SMX and GEN1 are restrained in S phase and become elevated late in the cell cycle to ensure the resolution of persistent recombination intermediates before mitotic division. By developing a "resolvase-deficient" model system in which the activities of MUS81 and GEN1 are compromised, we have explored the fate of unresolved recombination intermediates. We find that covalently linked sister chromatids promote the formation of a new class of ultrafine bridges at anaphase that we term HR-UFBs. These bridges are broken at cell division, leading to activation of the DNA damage checkpoint and repair by nonhomologous end joining (NHEJ) in the next cell cycle. As a consequence, high levels of gross chromosomal rearrangements and aberrations are observed, together with frequent cell death. These results show that the HJ resolvases provide essential functions for the resolution of recombination intermediates, even in cells that remain proficient for BTR-mediated HJ dissolution.

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 A-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.

Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation., The Journal of Cell Biology, 185 (5), ISSN:0021-9525, ISSN:1540-8140

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21CIP1/WAF1 were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21CIP1/WAF1, is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21CIP1/WAF1. These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.

DNA strand break repair by homologous recombination leads to the formation of intermediates in which sister chromatids are covalently linked. The efficient processing of these joint molecules, which often contain four-way structures known as Holliday junctions, is necessary for efficient chromosome segregation during mitotic division. Because persistent chromosome bridges pose a threat to genome stability, cells ensure the complete elimination of joint molecules through three independent pathways. These involve (1) BLM-Topoisomerase IIIα-RMI1-RMI2 (BTR complex), (2) SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and (3) GEN1. The BTR pathway promotes the dissolution of double Holliday junctions, which avoids the formation of crossover products, prevents sister chromatid exchanges, and limits the potential for loss of heterozygosity. In contrast to BTR, the other two pathways resolve Holliday junctions by nucleolytic cleavage to yield crossover and non-crossover products. To avoid competition with BTR, the resolution pathways are restrained until the late stages of the cell cycle. The temporal regulation of the dissolution/resolution pathways is therefore critical for crossover avoidance while also ensuring that all covalent links between chromosomes are resolved before chromosome segregation.

Holliday junction (HJ) resolvases are necessary for the processing of persistent recombination intermediates before cell division. Their actions, however, need to be restricted to the late stages of the cell cycle to avoid the inappropriate cleavage of replication intermediates. Control of the yeast HJ resolvase, Yen1, involves phosphorylation changes that modulate its catalytic activity and nuclear import. Here, we show that GEN1, the human ortholog of Yen1, is regulated by a different mechanism that is independent of phosphorylation. GEN1 is controlled exclusively by nuclear exclusion, driven by a nuclear export signal (NES) that restricts GEN1 actions to mitosis when the nuclear membrane breaks down. Construction of a nuclear-localized version of GEN1 revealed that its premature actions partially suppress phenotypes associated with loss of BLM and MUS81, but cause elevated crossover formation. The spatial control of GEN1 therefore contributes to genome stability, by avoiding competition with non-crossover promoting repair pathways., The human Holliday junction resolvase GEN1 functions during anaphase to eliminate recombination intermediates that block proper chromosome segregation. Here, the authors demonstrate that GEN1 activity is regulated independently of its phosphorylation status and relies on its active exclusion from the nucleus.

In January 2013, over 100 researchers from 18 different countries gathered in Obergurgl, a remote ski resort in the beautiful Tyrolean Alps in Austria. The inspiring idea of this EMBO-funded workshop was to bring together two scientific communities: researchers in the fields of cell cycle and cell death. These two mature fields have been considered distinct for many years and researchers active in one often tend to neglect aspects of the other. It is well established though that cell cycle defects can trigger cell death and it is becoming increasingly clear that some cell death paradigms can only (or preferentially) be found within certain cell cycle ‘windows'. Furthermore, defects in both pathways are hallmarks of cancer, amply justifying the concept of an interdisciplinary discussion platform. The quality of the talks and posters, the relaxed and informal atmosphere, enhanced by the beautiful weather and the stunning location, guaranteed the effectiveness of the interchange. Here we summarize a selection of the key contributions in the various fields and apologize to the participants neglected due to space constrains.

Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska recruitment to kinetochores and stabilization of kinetochore–microtubule attachments., The KMN network (named according to the acronym for KNL1, Mis12, and Ndc80) and the more recently identified Ska complex (Ska1–3) have been shown to mediate kinetochore (KT)–microtubule (MT) attachments. How these two complexes cooperate to achieve stable end-on attachments remains unknown. In this paper, we show that Aurora B negatively regulates the localization of the Ska complex to KTs and that recruitment of the Ska complex to KTs depends on the KMN network. We identified interactions between members of the KMN and Ska complexes and demonstrated that these interactions are regulated by Aurora B. Aurora B directly phosphorylated Ska1 and Ska3 in vitro, and expression of phosphomimetic mutants of Ska1 and Ska3 impaired Ska KT recruitment and formation of stable KT–MT fibers (K-fibers), disrupting mitotic progression. We propose that Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska recruitment to KTs and stabilization of KT–MT attachments.

Overriding the G(2) DNA damage checkpoint permits precocious entry into mitosis that ultimately leads to mitotic catastrophe. Mitotic catastrophe is manifested by an unscheduled activation of CDK1, caspase activation and apoptotic cell death. We found that although cyclin B1 was required for mitotic catastrophe, it was cleaved into a approximately 35 kDa protein by a caspase-dependent mechanism during the process. Cyclin B1 cleavage occurred after Asp123 in the motif ILVD(123) downward arrow, and mutation of this motif attenuated the cleavage. Cleavage was abolished by a pan-caspase inhibitor as well as by specific inhibitors for the effector caspase-6 and the initiator caspase-8. Cleavage created a truncated cyclin B1 lacking part of the NH(2)-terminal regulatory domain that included the destruction box sequence. Although cleavage of cyclin B1 itself was not absolutely required for mitotic catastrophe, expression of the truncated product enhanced cell death. In support of this, ectopic expression of this truncated cyclin B1 was not only sufficient to induce mitotic block and apoptosis but also enhanced mitotic catastrophe induced by ionizing radiation and caffeine. These data underscore a possible linkage between mitotic and apoptotic functions by caspase-dependent processing of mitotic activators.

Spindle-disrupting agents and CDK inhibitors are important cancer therapeutic agents. Spindle toxins activate the spindle-assembly checkpoint and lead to sustained activation of CDK1. Different published results indicate that CDK1 activity is either important or dispensable for the cytotoxicity associated with spindle disruption. Using live cell imaging and various approaches that uncoupled mitotic events, we show that apoptosis was induced by both prolonged nocodazole treatment as well as by inhibition of CDK1 activity after a transient nocodazole block. However, distinct mechanisms are involved in the two types of cell death. The massive apoptosis triggered by nocodazole treatment requires the continuous activation of cyclin B1-CDK1 and is antagonized by premature mitotic slippage. By contrast, apoptosis induced by nocodazole followed by CDK inhibitors occurred after rereplication and multipolar mitosis of the subsequent cell cycle. The presence of dual mechanisms of cytotoxicity mediated by spindle disruption and CDK inhibition may reconcile the various apparent inconsistent published results. These data underscore the essential role of cyclin B1-CDK1 as the basis of apoptosis during mitotic arrest, and the role of mitotic slippage and abnormal mitosis for apoptosis at later stages.

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.

The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. Here, we determine the fate of unresolved recombination intermediates arising in cells lacking two nucleases required for resolution (GEN1 -/- knockout cells depleted of MUS81). We find that intermediates persist until mitosis and form a distinct class of anaphase bridges, which we term homologous recombination ultra-fine bridges (HR-UFBs). HR-UFBs are distinct from replication stress-associated UFBs, which arise at common fragile sites, and from centromeric UFBs. HR-UFBs are processed by BLM helicase to generate single-stranded RPA-coated bridges that are broken during mitosis. In the next cell cycle, DNA breaks activate the DNA damage checkpoint response, and chromosome fusions arise by non-homologous end joining. Consequently, the cells undergo cell cycle delay and massive cell death. These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability.