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Key Points The spindle checkpoint signalling cascade prevents anaphase onset until all chromosomes are correctly attached, through their kinetochores, to spindle microtubules. Molecular interactions between kinetochore and spindle checkpoint proteins have been defined and characterized. There have been significant advances in understanding the molecular details of phosphoregulation and checkpoint scaffolding. Monopolar spindle protein 1 (MPS1) has emerged as a direct activating kinase of the checkpoint. The checkpoint response strength is variable and corresponds with the number of unattached kinetochores. Inactivation of cyclin-dependent kinase 1 (CDK1) by cyclin B degradation is a basis for checkpoint inactivation during anaphase. Nuclear pore complexes, in addition to kinetochores, signal the checkpoint. The mechanisms underlying spindle checkpoint signalling at the kinetochore, which ensures faithful chromosome segregation during cell division, are being unravelled. They indicate that the checkpoint response is graded rather than switch-like (completely on or off) as traditionally thought, and provide insights for the treatment of cancers in which the checkpoint is bypassed. The spindle checkpoint ensures proper chromosome segregation during cell division. Unravelling checkpoint signalling has been a long-standing challenge owing to the complexity of the structures and forces that regulate chromosome segregation. New reports have now substantially advanced our understanding of checkpoint signalling mechanisms at the kinetochore, the structure that connects microtubules and chromatin. In contrast to the traditional view of a binary checkpoint response — either completely on or off — new findings indicate that the checkpoint response strength is variable. This revised perspective provides insight into how checkpoint bypass can lead to aneuploidy and informs strategies to exploit these errors for cancer treatments.

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle. A key feature of living cells is the cell cycle. In this Perspective, the authors explore attempts to recreate this process and what is still required for an integrated synthetic cell cycle.

Key Points Replication fork reversal is a complex transaction that requires the coordinated unwinding and annealing of parental and newly synthesized strands at the replication fork. Replication fork reversal is a frequent transaction in mammalian cells following different types of genotoxic stress. However, it is usually disfavoured in yeast cells, where alternative events such as re-priming are particularly efficient, and key regulatory factors such as poly(ADP-ribose) polymerase (PARP) are absent. Fork reversal can be seen as a 'double-edged sword'. It contributes to genome stability by promoting DNA damage tolerance and repair, and by protecting chromosome integrity during replication; however, it can also lead to microsatellite instability and unscheduled chromosomal breakage. Various factors can drive fork reversal and fork restoration in vitro , but their activity heavily depends on the molecular features of the DNA substrate and on the presence of auxiliary factors. Fork reversal in vivo is dependent on the homologous recombination factor RAD51, and is regulated by PARP and ATP-dependent RNA helicase Q1 (RECQ1)-mediated fork restart, but additional — mostly unknown — factors are likely to have a role in this process. Fork reversal dynamics have intriguing implications for our understanding of basic replication mechanisms and the DNA damage response. The cellular factors modulating fork remodelling are potentially attractive targets for cancer therapy. Replication perturbation causes replication fork reversal (remodelling). Recent studies have visualized replication forks in metazoan cells and identified fork remodelling factors, showing fork reversal to be a global and regulated process with potential effects on replication termination, genome stability and the DNA damage response. The remodelling of replication forks into four-way junctions following replication perturbation, known as fork reversal, was hypothesized to promote DNA damage tolerance and repair during replication. Albeit conceptually attractive, for a long time fork reversal in vivo was found only in prokaryotes and specific yeast mutants, calling its evolutionary conservation and physiological relevance into question. Based on the recent visualization of replication forks in metazoans, fork reversal has emerged as a global, reversible and regulated process, with intriguing implications for replication completion, chromosome integrity and the DNA damage response. The study of the putative in vivo roles of recently identified eukaryotic factors in fork remodelling promises to shed new light on mechanisms of genome maintenance and to provide novel attractive targets for cancer therapy.

Key Points A living organism's genomic DNA, which is contained in each cell, is typically far longer than the organism itself. This poses a formidable challenge for DNA compaction in the cell nucleus and its segregation during cell division. Members of the structural maintenance of chromosomes (SMC) family are abundant and universal chromosomal protein components. They take on crucial roles in compacting and segregating both prokaryotic and eukaryotic genomes. SMC complexes are ring-shaped ATPases that bind to chromosomes by topological embrace. They are thought to structure and safeguard chromosomes by engaging in interactions between more than one fragment of DNA. They also recruit and interact with additional chromosomal proteins. The condensin complex may compact chromosomes by providing dynamic links between its binding sites, whereas cohesin has evolved special features to establish enduring links between newly replicated sister chromatids. Mutations in SMC complexes and their regulators are the cause of grave human malignancies, including cancer and developmental disorders. SMC (structural maintenance of chromosomes) complexes are found in all living organisms and include condensin, cohesin and the SMC5–SMC6 complex. Recent mechanistic insight into these ring-shaped protein machines, which topologically encircle DNA, shed light on how they function to mediate chromosome condensation, sister chromatid cohesion and DNA repair. SMC (structural maintenance of chromosomes) complexes — which include condensin, cohesin and the SMC5–SMC6 complex — are major components of chromosomes in all living organisms, from bacteria to humans. These ring-shaped protein machines, which are powered by ATP hydrolysis, topologically encircle DNA. With their ability to hold more than one strand of DNA together, SMC complexes control a plethora of chromosomal activities. Notable among these are chromosome condensation and sister chromatid cohesion. Moreover, SMC complexes have an important role in DNA repair. Recent mechanistic insight into the function and regulation of these universal chromosomal machines enables us to propose molecular models of chromosome structure, dynamics and function, illuminating one of the fundamental entities in biology.

Our understanding of the downstream effectors and upstream regulators of target of rapamycin (TOR) signalling continues to grow. In particular, recent global 'omics' studies have revealed physiological roles of mammalian TOR (mTOR) in protein, nucleotide and lipid synthesis, and other studies showed that Hippo, WNT and Notch signalling are novel regulators of mTOR. More than 20 years after its discovery, our understanding of target of rapamycin (TOR) signalling continues to grow. Recent global 'omics' studies have revealed physiological roles of mammalian TOR (mTOR) in protein, nucleotide and lipid synthesis. Furthermore, emerging evidence provides new insight into the control of mTOR by other pathways such as Hippo, WNT and Notch signalling. Together, this progress has expanded the list of downstream effectors and upstream regulators of mTOR signalling.

Key Points The chromosomal passenger complex (CPC) is a 'master controller' of cell division that is formed by a kinase module (Aurora B kinase) and a localization module (the scaffolding protein inner centromere protein (INCENP), survivin and borealin). Multiple post-translational modifications of CPC components contribute to the appropriate localization and regulation of Aurora B activity. Full activation of Aurora B kinase is a complex multistage process that is mediated by the other CPC components and other cell cycle kinases. In early mitosis, CPC recruitment to the inner centromere is mediated by post-translational modifications of two histones: phosphorylation of histone H3 (by haspin kinase) and of histone H2A (by Bub1 kinase). The baculovirus IAP repeat (BIR) domain of survivin recognizes H3 phosphorylated at Thr3. Further enrichment of the CPC at the inner centromere is mediated by Aurora B-dependent regulatory feedback loops. Roles of the CPC in early mitosis include the regulation of chromosome structure, kinetochore–microtubule attachments and the spindle assembly checkpoint. The CPC relocalizes to central spindle microtubules at the onset of anaphase in a highly regulated process that is mediated by a decrease of cyclin-dependent kinase 1 (Cdk1) activity, interaction with the kinesin mitotic kinesin-like protein 2 (Mklp2) and under the control of several phosphatases and Aurora B kinase itself. Functions of the CPC in late mitosis include the formation and stabilization of the spindle midzone in anaphase and the regulation of the contractile ring formation. The CPC has further roles later on in cytokinesis, in which it regulates furrow ingression and the abscission checkpoint. The chromosomal passenger complex (CPC), which is formed by inner centromere protein (INCENP), borealin, survivin and Aurora B kinase, targets to different locations at different times during mitosis. As it regulates key events at each of these locations, the CPC can be considered as a master regulator of mitosis. Successful cell division requires the precise and timely coordination of chromosomal, cytoskeletal and membrane trafficking events. These processes are regulated by the competing actions of protein kinases and phosphatases. Aurora B is one of the most intensively studied kinases. In conjunction with inner centromere protein (INCENP), borealin (also known as Dasra) and survivin it forms the chromosomal passenger complex (CPC). This complex targets to different locations at differing times during mitosis, where it regulates key mitotic events: correction of chromosome–microtubule attachment errors; activation of the spindle assembly checkpoint; and construction and regulation of the contractile apparatus that drives cytokinesis. Our growing understanding of the CPC has seen it develop from a mere passenger riding on the chromosomes to one of the main controllers of mitosis.

Key Points Meiotic recombination is a major source of genetic diversity in a population. Recent advances in mapping recombination hot spots have shed light on the evolutionary dynamics of recombination hotspot localization and on the factors involved in their specification. The localization of meiotic recombination sites in humans and mice is determined by the DNA-binding specificity of PR domain-containing 9 (PRDM9), which is instrumental in the specification of recombination hot spots. The PRDM9 DNA-binding domain has quickly evolved under positive selection. This evolution may be linked to the erosion of PRDM9-binding sites owing to meiotic DNA double-strand break (DSB) repair.. Meiotic DSBs are catalysed by the meiotic recombination protein SPO11 in humans and mice and are regulated by other proteins, which leads to the regulation of recombination. Several factors required for DSB formation are localized on chromosome axes, and this association with chromosome axes regulates meiotic recombination, from the formation of DSBs to their resolution into final recombination products that are formed from either crossover or non-crossover intermediates. In mammals, the proteins RING finger protein 212 (RNF212) and human enhancer of invasion 10 (HEI10) have a key role in promoting DSB repair towards crossovers from a subset of recombination intermediates. These findings provide new insights into the control of crossover frequency. Genetic exchanges between homologous chromosomes are essential for producing haploid gametes and increase genetic diversity. Recent advances have provided insights into the mechanisms that specify recombination hot spots and the regulation of the exchange of genetic material between mammalian chromosomes. During meiosis, a programmed induction of DNA double-strand breaks (DSBs) leads to the exchange of genetic material between homologous chromosomes. These exchanges increase genome diversity and are essential for proper chromosome segregation at the first meiotic division. Recent findings have highlighted an unexpected molecular control of the distribution of meiotic DSBs in mammals by a rapidly evolving gene, PR domain-containing 9 ( PRDM9 ), and genome-wide analyses have facilitated the characterization of meiotic DSB sites at unprecedented resolution. In addition, the identification of new players in DSB repair processes has allowed the delineation of recombination pathways that have two major outcomes, crossovers and non-crossovers, which have distinct mechanistic roles and consequences for genome evolution.

During prophase of the first meiotic division, cells deliberately break their DNA 1 . These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes 2 . A pathway that depends on the MLH1–MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism 3 – 7 . Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4–MSH5 (MutSγ), which supports crossing over 8 , binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ–MutSγ–EXO1–RFC–PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points 9 . As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC–PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes 4 . Reconstitution of the activation of the MLH1–MLH3 endonuclease shows how crossovers are formed during meiosis.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.Senescent cells have complex and important roles in cancer and ageing, but they are quite rare and difficult to characterize in tissues in vivo. In this Expert Recommendation, the SenNet Biomarkers Working Group discusses recent advances in detecting and characterizing cellular senescence and provides recommendations for senescence markers in 14 human and mouse tissues.