To fire or not to fire: origin activation in Saccharomyces cerevisiae ribosomal DNA.

Duplication of eukaryotic chromosomes begins from multiple sites called origins of DNA replication, with replication typically proceeding bidirectionally from each origin. The yeast Saccharomyces cerevisiae is the only eukaryote in which there is a detailed understanding of the sites used for initiation of chromosomal DNA replication. In yeast, these sites are called ARSs (autonomously replicating sequences) because they were initially identified by their ability to confer high-frequency transformation and self-replication on plasmids introduced into cells (Stinchcomb et al. 1979). By these criteria, there are 200–400 ARS elements in the yeast genome. Using two-dimensional (2D) gel electrophoresis, some—but not all—ARSs can be shown to be origins of replication when situated at their normal chromosomal loci (for review, see Newlon and Theis 2002). ARS elements are relatively small, ∼ 100–150 bp, and consist of one or more copies of an essential 11-bp-long AT-rich ARS consensus sequence, as well as several other less conserved elements. The ARS consensus sequence is the binding site for the multi-subunit origin recognition complex (ORC), which binds constitutively throughout the cell cycle and is essential for initiation (for review, see Bell and Dutta 2002). Several proteins are recruited to the ARS during the G1 phase of the cell cycle to form a prereplicative complex, including the multisubunit minichromosome maintenance (MCM) complex, which has ATPase and helicase activity and is needed for both initiation and fork progression. Recently, two laboratories used genome-wide microarray analysis to determine the positions of replication origins in the Saccharomyces genome (Raghuraman et al. 2001; Wyrick et al. 2001). Raghuraman and colleagues (2001) determined the time of replication of each segment of the 16 yeast chromosomes. They used this information to identify 332 sites where bidirectional replication begins, although many of these origins are not active in every cell cycle. Wyrick and colleagues (2001) used a complementary approach: They determined sites of binding of both the ORC and MCM complexes, finding 429 sites that are bound by both complexes. There is high but not complete overlap between the sites identified as origins by the two methods, suggesting that although ORC and MCM binding is highly correlated with origin activity, this binding is not sufficient and, in rare cases, may even be unnecessary for origin activity. Despite the wealth of information about initiation of DNA replication in Saccharomyces, it is still uncertain what determines whether a given yeast ARS acts as an origin or how the efficiency and time of activation of active origins is regulated (for review, see Pasero and Schwob 2000). Existing data suggest that both chromosomal context and chromatin structure affect origin activity. For example, if an origin is near a telomere, activation of this origin occurs late in S phase (Ferguson et al. 1991). If the same origin is placed on a plasmid, initiation occurs early in the cell cycle. However, linearization of this plasmid by addition of telomere sequences to both ends causes the origin to again be activated late (Ferguson and Fangman 1992). These data demonstrate that telomeres exert a position effect on the activation of nearby origins, a function reminiscent of their inhibitory effect on transcription of nearby genes (for review, see Tham and Zakian 2002). In some cases, chromosomal context actually prevents rather than simply delaying origin activation. For example, ARSs near HML, one of two transcriptionally quiescent mating type loci on chromosome III, and some origins in subtelomeric regions (Newlon et al. 1993; Ivessa et al. 2002) are typically inactive (Fig. 1). Although ARS301, which overlaps the location of the E silencer at HML, is not active in its normal chromosomal context, if it is moved away from HML to the position of and replacing ARS305, the first active origin on the left arm of chromosome III, ARS301 becomes active (Fig. 1; Vujcic et al. 1999). Further, the probability that a normally inactive ARS will become active increases the more time it takes a fork emanating from an active origin to reach the silent ARS. For example, in the HML region, if the active origin ARS305 is deleted, the ARS cluster 302, 303, 320 becomes active (Fig. 1). If the two active origins ARS305 and ARS306 are both absent, both ARS301 (at the E siCorresponding author. E-MAIL vzakian@molbio.princeton.edu; FAX (609) 258-1701. Article and publication are at http://www.genesdev. org/cgi/doi/10.1101/ gad.1033702.