Félix Machín

SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5–Smc6 complexes. The Smc5–Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5–Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5–Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.

Smc5–Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5–Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5–Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5–Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

Anaphase Onset Before Complete DNA Replication with Intact Checkpoint Responses

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most significantly at natural replication-impeding loci like the ribosomal DNA gene cluster. In the absence of Smc5-Smc6, chromosome nondisjunction occurs as a consequence of mitotic entry with unfinished replication despite intact checkpoint responses. Eliminating processes that obstruct replication fork progression restores the temporal uncoupling between replication and segregation in smc5-smc6 mutants. We propose that the completion of replication is not under the surveillance of known checkpoints.

Cdc14 inhibits transcription by RNA polymerase I during anaphase

Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.

Design and Synthesis of a Novel Series of Pyranonaphthoquinones as Topoisomerase II Catalytic Inhibitors

On the basis of previous pharmacophore modeling studies of naphthoquinones derivatives, we have designed and synthesized a new set of pyranonaphthoquinones. These compounds were obtained through a direct and highly efficient approach based on an intramolecular domino Knoevenagel hetero Diels-Alder reaction from lawsone (2-hydroxynaphthoquinone) and a variety of aldehydes containing an alkene. The synthesized pyranonaphthoquinones were evaluated against the alpha isoform of human topoisomerase II (hTopoIIalpha). Among the 11 derivatives studied, we found that six of them act as catalytic inhibitors of the enzyme in vitro. These six derivatives strongly preclude the enzyme from decatenating or relaxing suitable substrates. Finally, we correlate their active/inactive status with docking studies of these novel compounds into the ATPase domain of hTopoIIalpha.

Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.

Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network.

In order to transmit a full genetic complement cells must ensure that all chromosomes are accurately split and distributed during anaphase. Chromosome XII in S. cerevisiae contains the site of nucleolar assembly, a 1-2Mb array of rDNA genes named RDN1. Cdc14p is a conserved phosphatase, essential for anaphase progression and mitotic exit, which is kept inactive at the nucleolus until mitosis. In early anaphase, the FEAR network (Cdc Fourteen Early Anaphase Release) promotes the transient and partial release of Cdc14p from the nucleolus. The putative role of Cdc14p released by the FEAR network is thought to be the stimulation of full Cdc14p release by activation of the GTPase-driven signalling cascade (the Mitotic Exit Network or MEN) that ensures mitotic exit. Here, we show that nucleolar segregation is spatially separated and temporally delayed from the rest of the genome. Nucleolar segregation occurs during mid-anaphase and coincides with the FEAR release of Cdc14p. Inactivation of FEAR delays nucleolar segregation until late anaphase, demonstrating that one function of the FEAR network is to promote segregation of repetitive nucleolar chromatin during mid-anaphase. Links to supplemental material: http://www.landesbioscience.com/supplement/aragonCC3-4-sup.pdf http://www.landesbioscience.com/supplement/aragonCC3-4-supmov.mov

The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans.

We have characterized the CDC14 gene, which encodes a dual-specificity protein phosphatase in Candida albicans, and demonstrated that its deletion results in defects in cell separation, mitotic exit and morphogenesis. The C. albicans cdc14Δ mutants formed large aggregates of cells that resembled those found in ace2-null strains. In cdc14Δ cells, expression of Ace2p target genes was reduced and Ace2p did not accumulate specifically in daughter nuclei. Taken together, these results imply that Cdc14p is required for the activation and daughter-specific nuclear accumulation of Ace2p. Consistent with a role in cell separation, Cdc14p was targeted to the septum region during the M-G1 transition in yeast-form cells. Interestingly, hypha-inducing signals abolished the translocation of Cdc14p to the division plate, and this regulation depended on the cyclin Hgc1p, since hgc1Δ mutants were able to accumulate Cdc14p in the septum region of the germ tubes. In addition to its role in cytokinesis, Cdc14p regulated mitotic exit, since synchronous cultures of cdc14Δ cells exhibited a severe delay in the destruction of the mitotic cyclin Clb2p. Finally, deletion of CDC14 resulted in decreased invasion of solid agar medium and impaired true hyphal growth.

Transcription of ribosomal genes can cause nondisjunction.

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.

Smc5-Smc6 Complex Preserves Nucleolar Integrity in S. cerevisiae

As a baton in a relay race, intact genomes need to be smoothly passed onto daughter cells every cell generation. Cohesin and condensin are multiprotein complexes involved in chromosome segregation during mitosis, they perform the crucial function of organizing and compacting chromosomes into pairs to facilitate their equal distribution in anaphase. Both complexes share a core of similar origin, containing a heterodimer formed by members of the conserved chromosomal ATPase family named Smc. A third complex containing Smc proteins at its core, the Smc5-Smc6 complex, previously known to be involved in DNA repair has recently been shown to contribute to chromosome segregation during anaphase. Smc5-Smc6 plays a role in the disjunction of repetitive regions. Here, we present results further supporting the importance of Smc5-Smc6 in maintaining the integrity of the repetitive ribosomal DNA (rDNA) locus, the largest repetitive region of the budding yeast genome.