The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In C. elegans, PCM assembly requires the coiled-coil protein SPD-5. Here we found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle-and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.Genetic inheritance requires exceptional genetic stability over many generations of cells and organisms. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell cycle machinery with a series of surveillance pathways, termed checkpoints, that provide the cells with the capacity to survive genotoxic insults. These protective mechanisms are signal transduction pathways specialized in detecting abnormal DNA structures and in coordinating cell cycle progression with DNA repair. Their activation leads to cell cycle progression delay and concomitant activation of DNA repair pathways, thus preventing replication or segregation of damaged DNA molecules.The keystone of the DNA damage checkpoint is a protein kinase family related to phosphoinositide 3-kinase, among which are Saccharomyces cerevisiae Mec1 (48,61,90) and Tel1 (26, 52), Schizosaccharomyces pombe Rad3 (5), Drosophila melanogaster Mei-41 (29), and mammalian ATR (5) and ATM (72). These protein kinases respond to various stresses by phosphorylating key proteins, thus regulating numerous processes, depending on the spectra of their substrates (for reviews, see references 1 and 75). In particular, S. cerevisiae Mec1 and S. pombe Rad3, more closely related to human ATR, are the prototype transducers of the DNA damage and replication stress signals; they respond to UV damage, double-strand breaks (DSBs), and stalled replication forks. Conversely, yeast Tel1, similar to human ATM, is likely involved only in the response to DSBs (for reviews, see references 57 and 75).Tel1 and Mec1 also contribut...

SUMMARY Assembly of SAS-6 dimers to form the centriolar cartwheel requires the ZYG-1/Plk4 kinase. Here we show that ZYG-1 recruits SAS-6 to the mother centriole independently of its kinase activity; kinase activity is subsequently required for cartwheel assembly. We identify a direct interaction between ZYG-1 and the SAS-6 coiled-coil that explains its kinase activity-independent function in SAS-6 recruitment. Perturbing this interaction, or the interaction between an adjacent segment of the SAS-6 coiled-coil and SAS-5, prevented SAS-6 recruitment and cartwheel assembly. SAS-6 mutants with alanine substitutions in a previously described ZYG-1 target site or in 37 other residues, either phosphorylated by ZYG-1 in vitro or conserved in closely related nematodes, all supported cartwheel assembly. We propose that ZYG-1 binding to the SAS-6 coiled-coil recruits the SAS-6—SAS-5 complex to the mother centriole, where a ZYG-1 kinase activity-dependent step, whose target is unlikely to be SAS-6, triggers cartwheel assembly.