Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion. INTRODUCTIONThe maintenance of proper ploidy during cell division requires both the accurate replication of chromosomes and their faithful segregation during mitosis. The physical association of sister chromatids after DNA replication, or sister chromatid cohesion, is crucial for the proper segregation of sister chromatids at anaphase and is therefore critical for genome stability. In Saccharomyces cerevisiae, cohesion is mediated by a multisubunit protein complex called cohesin that is composed of at least four proteins: Smc1p, Smc3p, Mcd1p/Scc1p, and Irr1p/Scc3p (SA1 or SA2 in mammalian cells) (Guacci et al., 1997;Michaelis et al., 1997;Toth et al., 1999). Pds5p is also required for sister chromatid cohesion and its localization to chromatin requires Mcd1p/Scc1p (Hartman et al., 2000;Panizza et al., 2000). Before the onset of anaphase, Esp1p, a protease required for the separation of sister chromatids, is bound to its inhibitor Pds1p (Ciosk et al., 1998). At the onset of anaphase Pds1p is ubiquitinated and targeted for degradation by the anaphase promoting complex/cyclosome (APC/C) (Cohen-Fix et al., 1996). Degradation of Pds1p releases Esp1p, which then cleaves the cohesin subunit Scc1p resulting in sister chromatid separation (Uhlmann et al., , 2000.Other proteins required for proper sister chromatid cohesion function during the establishment of cohesion, which takes place during S phase. Scc2p and Scc4p physically interact with each other but are not core components of the cohesin complex (Ciosk et al., 2000). Scc2p and Scc4p are, however, required for the association of cohesin with DNA (Ciosk et al., 2000). Eco1p/Ctf7p is also required for the establishment but not the maintenance of cohesion (Skibbens et al., 1999;Toth et al., 1999). In eco1 mutants, the cohesin complex is able to associate with DNA, but proper sister chromatid cohesion is not establi...

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an ␣-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1⌬N301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1⌬N301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1⌬N301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1⌬N300 dimer, confirming that the latter represents a domain-swapped species.15 N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ϳ15-and ϳ75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.Gene expression can be controlled by modulating the DNA binding affinity of sequence specific transcription factors. Similar to several other transcription factors, the DNA binding of Ets-1 is modulated by an autoinhibitory module that provides a route to biological regulation (1). The Ets-1 inhibitory module is composed of sequences flanking the winged helix-turn-helix (HTH) 1 DNA binding ETS domain (2, 3). When these sequences are deleted, as in an alternatively spliced isoform of Ets-1, or when their structural elements are disrupted by mutations, as in the case of the oncogenic v-Ets, the affinity of Ets-1 for its target DNA sites is enhanced by 10-to 20-fold (4 -6). In a cellular context, this module is essential for response to different regulatory signals. DNA binding of Ets-1 is enhanced 10-to 20-fold through a partnership with the transcription factor RUNX1 (CBF␣2/AML1) (7). Conversely, in activated T-cells, phosphorylation of a serine-rich region (residues 244 -300) inhibits the DNA binding of Ets-1 by another ϳ50-fold (8). Importantly, these two effects require an intact inhibitory module.Mechanistic insight into autoinhibition has come from the observation that the Ets-1 inhibitory module changes conformation upon binding to DNA. Initial secondary structural studies performed in our laboratories demonstrated that this module is composed of four coupled ␣-helices, locat...

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Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.chromosome stability ͉ synthetic genetic array ͉ kinetochore