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...

We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.

Here we demonstrate that open reading frame 16 (ORF16) of the oncogenic herpesvirus saimiri protects cells from heterologous virus-induced apoptosis. The BH1 and BH2 homology domains are highly conserved in ORF16, and ORF16 heterodimerizes with Bcl-2 family members Bax and Bak. However, ORF16 lacks the core sequence of the conserved BH3 homology domain, suggesting that this region is not essential for anti-apoptotic activity. Conservation of a functional bcl-2 homolog among gammaherpesviruses suggests that inhibition of programmed cell death is important in the biology of these viruses.

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