Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction

The SUMO Isopeptidase Ulp2p Is Required to Prevent Recombination-Induced Chromosome Segregation Lethality following DNA Replication Stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress

Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance

Sumoylation represents a conserved mechanism of post-translational protein modification. We report that Pli1p, the unique fission yeast member of the SP-RING family, is a SUMO E3 ligase in vivo and in vitro. pli1Δ cells display no obvious mitotic growth defects, but are sensitive to the microtubule-destabilizing drug TBZ and exhibit enhanced minichromosome loss. The weakened centromeric function of pli1Δ cells may be related to the defective heterochromatin structure at the central core, as shown by the reduced silencing of an ura4 variegation reporter gene inserted at cnt and imr. Interestingly, pli1Δ cells also exhibit enhanced loss of the ura4 reporter at these loci, likely by gene conversion using homologous sequences as information donors. Moreover, pli1Δ cells exhibit consistent telomere length increase, possibly achieved by a similar process. Point mutations within the RING finger of Pli1p totally or partially reproduce the pli1 deletion phenotypes, thus correlating with their sumoylation activity. Altogether, these results strongly suggest that Pli1p, and by extension sumoylation, is involved in mechanisms that regulate recombination in particular heterochromatic repeated sequences

Reconstitution and subunit geometry of human condensin complexes

Vertebrate cells possess two different condensin complexes, known as condensin I and condensin II, that play a fundamental role in chromosome assembly and segregation during mitosis. Each complex contains a pair of structural maintenance of chromosomes (SMC) ATPases, a kleisin subunit and two HEAT-repeat subunits. Here we use recombinant human condensin subunits to determine their geometry within each complex. We show that both condensin I and condensin II have a pseudo-symmetrical structure, in which the N-terminal half of kleisin links the first HEAT subunit to SMC2, whereas its C-terminal half links the second HEAT subunit to SMC4. No direct interactions are detectable between the SMC dimer and the HEAT subunits, indicating that the kleisin subunit acts as the linchpin in holocomplex assembly. ATP has little, if any, effects on the assembly and integrity of condensin. Cleavage pattern of SMC2 by limited proteolysis is changed upon its binding to ATP or DNA. Our results shed new light on the architecture and dynamics of this highly elaborate machinery designed for chromosome assembly

Cdc14 phosphatase resolves the rDNA segregation delay

Sister chromatid segregation in anaphase of mitosis is initiated through cleavage of cohesin by the protease separase. Two studies now show that this view is valid for most chromosomal DNA, but not for the highly repetitive ribosomal DNA (rDNA) and telomeres. The disjunction of these regions of the chromosome occurs in mid-anaphase, long after cohesin cleavage, and is regulated by the conserved phosphatase Cdc14