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 Plk4 family kinases control centriole assembly. Plk4s target to mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular β-sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a new crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly.

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.

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Telomere structure allows cells to distinguish the natural chromosome ends from double-strand breaks (DSBs). However, DNA damage response proteins are intimately involved in telomere metabolism, suggesting that functional telomeres may be recognized as DNA damage during a time window. Here we show by two different systems that short telomeres are recognized as DSBs during the time of their replication, because they induce a transient MRX-dependent DNA damage checkpoint response during their prolonged elongation. The MRX complex, which is recruited at telomeres under these conditions, dissociates from telomeres concomitantly with checkpoint switch off when telomeres reach a new equilibrium length. We also show that MRX recruitment to telomeres is sufficient to activate the checkpoint independently of telomere elongation. We propose that MRX can signal checkpoint activation by binding to short telomeres only when they become competent for elongation. Because full-length telomeres are refractory to MRX binding and the shortest telomeres are elongated of only a few base pairs per generation, this limitation may prevent unscheduled checkpoint activation during an unperturbed S phase.

Although cytoplasmic interactions between Caenorhabditis elegans centrosome proteins SPD-5, RSA-1, and RSA-2 can be detected in vivo, the key proteins required for centrosome assembly, SPD-2 and SPD-5, exist mostly as monomers and are separated from the centrosome-related kinases PLK-1 and AIR-1 in the cytoplasm.

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition. INTRODUCTIONThe ends of eukaryotic chromosomes are protected by specialized functional complexes called telomeres from being recognized and processed as double-strand breaks (DSBs) (McClintock, 1941(McClintock, , 1942. The critical distinction between telomeres and DSBs is that the natural ends of linear chromosomes do not undergo end-to-end fusions and other processing events normally required to promote repair of DNA breaks. In addition, the presence of DSBs triggers the activation of surveillance mechanisms, known as DNA damage checkpoints, which ensure that damaged DNA molecules are neither replicated nor segregated to daughter cells until repaired (reviewed in Longhese et al., 1998;Zhou and Elledge, 2000;Kolodner et al., 2002;Nyberg et al., 2002;Rouse and Jackson, 2002).In most organisms, such as yeast and vertebrates, the ends of linear chromosomes contain telomeric DNA repeats of 5-25 base pairs that vary in sequence among species and terminate in a 3Ј single-stranded overhang (Wellinger and Sen, 1997), which is likely important for both recruitment of the telomerase enzyme and for efficient telomeric sequence addition. Telomeric DNA is usually maintained by the action of telomerase, a specialized reverse transcriptase that uses its RNA component as a template to elongate the TGrich strand of telomeric DNA (reviewed in Greider, 1996;Zakian, 1996). Telomerase-mediated lengthening of chromosomal ends in budding yeast involves a number of factors. In fact, the action of the Est2 catalytic reverse transcriptase protein subunit and of the TLC1 telomerase RNA is modulated by both the TLC1-binding proteins Est1 and Est3 and the single-stranded...