Separable Functions of ORC 5 in Replication Initiation and Silencing in Saccharomyces c

Origin recognition complex (ORC) is a six subunit complex that functions as the replication initiator and is required for silencing the HML and Hh4R loci in the yeast Saccharomyces cereuisiae. The roles of ORC5 in replication initiation and silencing were investigated to determine whether the two roles were mechanistically coincident or separable. Some spontaneous revertants of mc5-I were functional for replication initiation, but not silencing. Other alleles of ORC5 were obtained that were nonfunctional for replication initiation, but fully competent for silencing. The two types of alleles, when put in the same cell, complemented, establishing two separable functions for ORC5. These data implied that replication initiation at HMR" was not required for silencing. The data were consistent with a model in which different ORC species functioned at different origins within the genome and that only one Orc5p subunit functioned at any given origin. T HE origin recognition complex (ORC) is a six polypeptide complex that binds to the ARS consensus sequence found at all known origins of replication in the yeast Saccharomyces cerevisiae. Temperature-sensitive (ts) mutations in ORCgenes block replication initiation at origins of replication at the restrictive temperature and, in the case of orc2-1, cause cells to arrest in the cell cycle at the G1/S boundary with unreplicated DNA (BELL et al. 1993; Fox et al. 1995; LIANG et al. 1995; Loo et al. 1995). In vivo, mutations in ORC2 and ORC5, which encode the second and fifth largest ORC subunit, respectively, are defective in replication initiation at several origins of replication, including the origin found at the HMR-E silencer (Fox et al. 1995). Mutations in ORC genes derepress the mating type genes at HML and HMR, implying a link between DNA replication and silencing (FOSS et al. 1993; LOO et al. 1995). Silencing is a form of gene regulation that involves the assembly of certain regions of the genome into an inactive chromatin structure, which blocks gene expression. In S. cerevisiae, silencing blocks expression of the mating type genes at HML and HMR, and blocks gene expression at telomeres (RINE and HERSKOWITZ 1987; GOTTSCHLING et al. 1990). The silent domain of HMR is inaccessible to nucleases, RNA polymerases, and DNA methylases (SCHNELL and &NE 1986; SINGH and KLAR 1992; LOO and &NE 1994). In addition to ORC, silencing depends upon regulatory sites, known as silencers, which flank HML and HMR, the silencer binding proteins Raplp and Abflp and the four SIR genes (reviewed in LOO and RINE 1995). Moreover, mutations h s p o n d i n g author: Jasper Rine, Division of Genetics, 401 Barker Hall, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. E-mail: jasperQmendel.berkeley.edu in the genes encoding either histone H3 or histone H4 cause derepression of both HMR and HML loci, indicating that silencing involves an inactive structure of chromatin ( U Y N E et al. 1988; JOHNSON et al. 1990; PARK and SZOSTAK 1990; THOMPSON et at. 1994). Both HML and Hh4R are flanked by a pair of silencers, with the E silencer on the left and the Zsilencer on the right of both loci. Hh4R-E, the most thoroughly studied of the four silencers, contains three functional domains. Two of the domains correspond to binding sites for two well studied transcription factors: Abflp and Raplp. The third domain corresponds to a binding site for ORC. A synthetic silencer composed of only these three domains is fully capable of silencing expression of flanking genes ( MCNALLY and RINE 1991). Both the E and Z silencers at HMR are bona fide origins of replication, suggesting a link between DNA replication and silencing (RMER and RINE 1992; D. RMER and J. &NE, unpublished data; reviewed in DIL LIN and RINE 1995). However, several observations already hint that the relationship between DNA replication initiation and silencing is not simple. First, ORC is required outside of S phase for silencing, implying that its role in silencing is not restricted to ORC's role in replication initiation (FOX et al. 1995). Second, tethering Sirlp to synthetic silencers bypasses the need for ORC in silencing (TRIOLO and STERNGIANZ 1996; Fox et al. 1997). Third, expression of the ORC2 gene of Drosophila melanogaster in a yeast orc2-1 mutant complements the silencing defect but not the replication defect of orc2-1 (EHRENHOFER-MURRAY et al. 1999). Finally, like orc2-1, the 0x5-1 mutation decreases initiation of replication at A R S l , a bona fide origin of replication (LIANG et al. 1995). However, unlike orc2-1, which causes a simiGenetics 147: 1053-1062 (November, 1997) 1054 A. Dillin and J. Rine