SUMMARY Proper bipolar attachment of sister kinetochores to the mitotic spindle is critical for accurate chromosome segregation in mitosis. Here we show an essential role of the formin mDia3 in achieving metaphase chromosome alignment. This function is independent of mDia3 actin nucleation activity, but is attributable to EB1-binding by mDia3. Furthermore, the microtubule binding FH2 domain of mDia3 is phosphorylated by Aurora B kinase in vitro, and cells expressing the nonphosphorylatable mDia3 mutant cannot position chromosomes at the metaphase plate. Purified recombinant mDia3 phosphorylated by Aurora B exhibits reduced ability to bind microtubules and stabilize microtubules against cold-induced disassembly in vitro. Cells expressing the phosphomimetic mDia3 mutant do not form stable kinetochore microtubule fibers; despite they are able to congress chromosomes to the metaphase plate. These findings reveal a key role for mDia3 and its regulation by Aurora B phosphorylation in achieving proper stable kinetochore microtubule attachment.

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.

Fragile sites are classified as common or rare depending on their occurrence in the populations. While rare sites are mainly associated with inherited diseases, common sites have been involved in somatic rearrangements found in the chromosomes of cancer cells. Here we study a mouse locus containing the ionotropic glutamate receptor delta 2 (grid2) gene in which spontaneous chromosome rearrangements occur frequently, giving rise to mutant animals in inbred populations. We identify and clone common fragile sites overlapping the mouse grid2 gene and its human ortholog GRID2, lying respectively at bands 6C1 and 4q22 in a 7-Mb-long region of synteny. These results show a third example of orthologous common sites conserved at the molecular level, and reveal an unexpected link between an inherited disease and an aphidicolin-sensitive region. Recurrent deletions of subregions of band 4q22 have been previously described in human hepatocellular carcinomas. This 15-Mb-long region appears precisely centered on the site described here, which strongly suggests that it also plays a specific role in hepatic carcinogenesis.

Following prolonged mitotic spindle disruption by microtubule poisons, mammalian cells delay their entry into anaphase, then progressively slip out of mitosis and become tetraploid. Normal cells then stop cycling before S-phase onset, but the mechanisms underlying this arrest are still unclear. Here we show that a double block prevents endo-reduplication. First, cells that exit mitosis without a functional microtubule network are driven toward G0. Reconstitution of the network unmasks a second block that relies on DNA double-strand breaks occurring early in the G1 phase that follows the mitotic block. We propose that a stress signal elicited upon mitotic impairment triggers breakage, which couples the leaky spindle checkpoint to the stringent DNA damage response. Consistent with this finding, cells defective for the damage response continue cycling and acquire, within a single cell cycle, both chromosome rearrangements and abnormal chromosome numbers that remarkably mimic the complex genetic hallmark of tumorigenesis.

Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceIinduced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.

Summary Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association.

The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.