We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2–Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1–Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled‐coil to form two connected sub‐complexes with Mad2. The Mad2 C‐terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular ‘safety belts’. We show that Mad1 is a competitive inhibitor of the Mad2–Cdc20 complex, and propose that the Mad1–Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1–Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1–Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1–Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C‐terminal tail is required for ligand release from Mad2, and that the ‘safety belt’ mechanism may prolong the lifetime of Mad2–ligand complexes.

SummaryBrca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Summary Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as inner and outer kinetochore, respectively [reviewed in 1]. The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multi-subunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization [2–10]. Here, we show that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome mis-segregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and we observe checkpoint impairment in cells expressing the Cenp-C N-terminal segment. Our studies unveil a crucial and likely universal link between the inner and outer kinetochore.

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.DOI: http://dx.doi.org/10.7554/eLife.02978.001

Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.

Edited by Wilhelm JustCoordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.Keywords: DNA recombination; DNA replication; genome stability BRCA1, BRCA2, RAD51 and the RAD51 paralogs family, which consists of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) in mammalian cells, are required to repair DNA damage by homologous recombination (HR). Mutations in most of these genes predispose to cancer, indicating an important role of DNA damage repair in preventing cell transformation. Intriguingly, complete loss of function of most of HR proteins is incompatible with life in vertebrate organisms [1,2,3]. These features together with their ability to form foci in unperturbed and challenged S-phase nuclei indicate a role for HR proteins in chromosomal DNA replication even in unchallenged conditions [4,5].The mechanisms underlying the function of DNA repair proteins in unchallenged chromosomal DNA replication are poorly understood. This is in part due to the fact that many of the genes involved in DNA metabolism are essential for cell viability, which complicate their study, especially in higher eukaryotes [1,2,3]. The reasons why HR genes are essential for cell viability in higher eukaryotes are unclear. One explanation might be that they have a specific and direct role in the replication of complex eukaryotic genomes. Alternatively, complex genomes might be more vulnerable to spontaneous DNA damage, which might irreversibly halt replication progression inducing chromosomes breakage. HR factors might be therefore required to repair the damage and to complete whole genome duplication. Here we review the links between HR proteins and the DNA replication machinery, some of which appear to be conserved between prokaryotes and eukaryotes.

A chemical biology study characterizes the role of Haspin kinase in centromere recruitment of the chromosome passenger complex and in spindle assembly checkpoint function.