Abstract:

The Origin Recognition Complex (ORC) is a conserved six-subunit protein complex required for initiation of DNA replication in all eukaryotes, as well as transcriptional silencing at the silent-mating type loci in the budding yeast, Saccharomyces cerevisiae (Bell et al. , 1993; Foss et al. , 1993; Micklem et al. , 1993). In budding yeast, conditionally lethal orc mutations disrupt ORC-DNA interactions (Santocanale and Diffley, 1996) and cause multiple phenotypes. These include defects in replication initiation, silencing at sensitized HM silencers and at synthetic telomeres (Fox et al. , 1995a; Loo et al. , 1995), and infra-S phase checkpoints (Shirahige et al. , 1998a; Weinberger et al. , 1999).

A role for budding yeast ORC in transcriptional repression outside the silent mating type loci has been demonstrated for sequences at the 2? plasmid origin of replication (Grunweller and Ehrenhofer-Murray, 2002). Our results from the analysis of global changes in expression profiles of temperature-sensitive orc2-1 cells relative to isogenic wild type cells at the restrictive temperature suggest a wider role for ORC in transcriptional repression, mainly of genes induced by starvation and other stresses. These genes very often physically cluster and map near ORC-binding loci more frequently than expected and overlap significantly with genes and/or gene clusters induced by inactivation of silencing proteins. We have definitively established that in the absence of starvation conditions, ORC represses at least two genes in the DAL gene cluster on chromosome IX, harboring genes coordinately induced under starvation conditions. Analysis of another genomic locus on chromosome XIII, harboring genes induced by the orc2-1 mutation as well as by starvation, also strongly suggests that repression of these genes in the absence of starvation is likely mediated by ORC.

Genes repressed by ORC in the absence of starvation/stress include rapidly evolving paralogues that have acquired new, but less essential functions since an ancient whole genome duplication event. The slowly evolving paralogues of these genes are often associated with more essential functions required for growth or rapid responses to stress that require their constitutive expression in cycling cells. These genes are therefore less likely to be repressed in the absence of stress, and, consistent with this, they infrequently coincide with ORC-binding loci or silent genomic domains in cycling cells. Our results provide a rationale for the non-random organization of the budding yeast genome during evolution, which is the clustering of stress-induced genes in regions of the genome maintained silent by ORC and other proteins in the absence of stress, and the evolutionary exclusion of genes essential for viability and growth from these silent domains, to ensure reproductive and evolutionary fitness. (Abstract shortened by UMI.)