Centromeric (CEN) chromatin is placed under mechanical tension and stretches as kinetochores biorient on the mitotic spindle. This deformation could conceivably provide a readout of biorientation to error correction mechanisms that monitor kinetochore-spindle interactions, but whether CEN chromatin acts in a tensiometer capacity is unresolved. Here, we report observations linking yeast Topoisomerase II (Top2) to both CEN mechanics and assessment of interkinetochore tension. First, in top2-4 and sumoylation-resistant top2-SNM mutants CEN chromatin stretches extensively during biorientation, resulting in increased sister kinetochore separation and preanaphase spindle extension. Our data indicate increased CEN stretching corresponds with alterations to CEN topology induced in response to tension. Second, Top2 potentiates aspects of the tension checkpoint. Mutations affecting the Mtw1 kinetochore protein activate Ipl1 kinase to detach kinetochores and induce spindle checkpoint arrest. In mtw1top2-4 and mtw1top2-SNM mutants, however, kinetochores are resistant to detachment and checkpoint arrest is attenuated. For top2-SNM cells, CEN stretching and checkpoint attenuation occur even in the absence of catenation linking sister chromatids. In sum, Top2 seems to play a novel role in CEN compaction that is distinct from decatenation. Perturbations to this function may allow weakened kinetochores to stretch CENs in a manner that mimics tension or evades Ipl1 surveillance. INTRODUCTIONAccurate chromosome segregation requires that sister kinetochores connect to microtubules from opposing poles of the mitotic spindle, a process known as biorientation. Misaligned kinetochore attachments can also occur, and they must be resolved to prevent lethal chromosome segregation errors or the formation of aneuploid cells. Syntelic attachments arise when sister kinetochores connect to microtubules from the same spindle pole; merotelic attachments result if a single kinetochore attaches to both poles. The Aurora-B kinases, complexed with conserved INCENP/ Sli15 and Survivin/Bir1 subunits of the chromosomal passenger complex, are key regulators of the error correction machinery that resolves such inappropriate attachments (reviewed in Ruchaud et al., 2007). Aurora-B (Ipl1 in budding yeast) facilitates error correction through two mechanisms. First, these kinases destabilize defective kinetochore-microtubule interactions by phosphorylating kinetochore-associated proteins. Second, Ipl1/Aurora-B plays a role in activating the spindle assembly checkpoint (SAC), both in response to syntelic attachments as well as mutations that affect the kinetochore or sister chromatid cohesion. Checkpoint activation may occur indirectly because Ipl1/Aurora-B creates unoccupied kinetochores (Pinsky et al., 2006) or directly through phosphorylation of SAC regulators (King et al., 2007).The manner in which Ipl1/Aurora-B distinguishes such a wide range of spindle lesions is not well understood. With the exception of merotelic interactions (Cimini, 2007), those att...

Increased mitotic recombination enhances the risk for loss of heterozygosity, which contributes to the generation of cancer in humans. Defective DNA replication can result in elevated levels of recombination as well as mutagenesis and chromosome loss. In the yeast Saccharomyces cerevisiae, a null allele of the RAD27 gene, which encodes a structure-specific nuclease involved in Okazaki fragment processing, stimulates mutation and homologous recombination. Similarly, rad3-102, an allele of the gene RAD3, which encodes an essential helicase subunit of the core TFIIH transcription initiation and DNA repairosome complexes confers a hyper-recombinagenic and hypermutagenic phenotype. Combining the rad27 null allele with rad3-102 dramatically stimulated interhomolog recombination and chromosome loss but did not affect unequal sister-chromatid recombination, direct-repeat recombination, or mutation. Interestingly, the percentage of cells with Rad52-YFP foci also increased in the double-mutant haploids, suggesting that rad3-102 may increase lesions that elicit a response by the recombination machinery or, alternatively, stabilize recombinagenic lesions generated by DNA replication failure. This net increase in lesions led to a synthetic growth defect in haploids that is relieved in diploids, consistent with rad3-102 stimulating the generation and rescue of collapsed replication forks by recombination between homologs.

In vertebrate cells, the small ubiquitin-like modifier SUMO plays a poorly defined role in targeting DNA topoisomerase II (TopoII) to centromeres (CENs) during mitosis, presumably to facilitate the untangling of sister chromatids as cells transition into anaphase. A new study by Dawlaty in the April 4 issue of Cell identifies the nucleoporin RanBP2 as a novel tumor suppressor that acts as a SUMO ligase for TopoII. Analysis of this interaction reveals TopoII recruitment to CENs is likely to play an important role in preventing chromosome segregation errors that lead to cancer.

The ability to make specific genomic alterations is an invaluable tool to researchers who use genetics and biochemistry to study problems in biology. We have investigated some of the parameters governing DNA fragment transplacement in two commonly used strains of Saccharomyces cerevisiae, S288C and W303-1A. These strains exhibited a marked difference in their capacity to take up plasmid DNA and utilize linear DNA fragments as substrates for transplacement. The contributions of transformation efficiency, length of homology, and alternative target site configuration were assessed. This analysis indicates that several genetic parameters are important for optimizing the efficiency of gene transplacement.