Analysis of Chromosome III Replicators Reveals an Unusual Structure for the ARS318 Silencer Origin and a Conserved WTW Sequence within the Origin Recognition Complex Binding Site

Abstract

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.Chromosomal origins of DNA replication in budding yeast are called autonomously replicating sequence (ARS) elements and were identified about 30 years ago by their ability to confer autonomous replication to originless plasmids (30). A conserved 11-bp sequence called the ARS consensus sequence (ACS) was initially identified by the analysis of the DNA sequences of four ARS elements (8). The ACS is now known to comprise a binding site for the origin recognition complex (ORC), the essential initiator protein in all eukaryotes (reviewed in reference 2). The consensus sequence of the AT-rich ACS element (WTTTAYRTTTW) is degenerate, and bona fide ARSs sometimes contain only a 10/11-or 9/11-bp match to this sequence. A W indicates the base A or T. When additional ACSs were identified experimentally, a 17-bp extended ACS (EACS) was defined and reflected that the bases flanking the ACS were often A's or T's (WWW-ACS-[G/T]WW) (47). Although the ACS is essential for replicator activity, it cannot be the sole determinant of ORC binding and/or origin specification. By pattern matching, there are 860 exact matches to the 11-bp ACS and 13,978 ACSs, allowing one mismatch in the yeast nuclear genome (http://seq.yeastgenome.org/cgi-bin /PATMATCH/nph-patmatch). Since there are only ϳ350 functional ARS elements in Saccharomyces cerevisiae, excluding the ribosomal DNA locus (25), additional sequences, chromatin environment, active transcription units, higher-order nucleosome structure, and/or nuclear organization might further restrict the locations of functional ARS elements. In agreement with one of these predictions, genome-wide studies of ORC and MCM binding sites revealed that Ͼ90% of putative ARSs are intergenic (50, 51). Furthermore, additional sequences in the B region adjacent to the ACS (Fig. 1) are known to contribute to ORC binding and origin activity in S. cerevisiae (26,37,41,46).Detailed analysis of ARS1 (26) and ARS307 (37, 46) showed that they have modular structures (Fig. 1). In addition to the A...

SubtelomericACS-containing Proto-silencers Act as Antisilencers in Replication Factors Mutants inSaccharomyces cerevisiae

“…We see no such overlapping near-ACS matches at the telomere whereas the individual near-ACS matches are eight of 11 at best. At the same time, mutation of the B1 site next to the 11/11 ACS match did not have strong effect on the replication activity of HMR-E (ARS317) and the subtelomeric ARS319 (Chang et al, 2008) but have profound effect on silencing at HMR-E (Palacios DeBeer et al, 2003) and at the telomere (our findings). These observations provide circumstantial support to the notion that the B1/ACS element rather than near-ACS matches are responsible for the silencing effects at these positions.…”

“…At least two studies have reported functional dissimilarities between different B1 elements that relate to the replicator or silencer efficiency (Palacios DeBeer and Fox, 1999;Palacios DeBeer et al, 2003;Chang et al, 2008). We therefore asked whether the apparent differences of ACS in ARS1 and core X could be extended to their B1 elements.…”

“…A very recent article (Chang et al, 2008) mapped the essential replication activity of HMR-E to two overlapping 9/11 ACS matches, but not to the 11/11 ACS match of the silencer (ARS317). Is it then possible that the antisilencing activity at telomeres is mediated by near-ACS matches?…”

“…Finally, in orc2-1 and orc5-1, we ( Figure 2) and others (Palacios DeBeer and Fox, 1999;Pryde and Louis, 1999;Lebrun et al, 2001;Fourel et al, 2002b) have observed very strong de-repression uncharacteristic for other mutants. Hence, we do not exclude the possibility that the strong effects in Figure 2 are consequence of the combination of the gene mutations and the ablation of ACS and do not represent ACS-driven effects only.A very recent article (Chang et al, 2008) mapped the essential replication activity of HMR-E to two overlapping 9/11 ACS matches, but not to the 11/11 ACS match of the silencer (ARS317). Is it then possible that the antisilencing activity at telomeres is mediated by near-ACS matches?…”

Conservation of replication timing reveals global and local regulation of replication origin activity

“…Comparative genomic approaches in the sensu stricto group of Saccharomyces yeasts revealed phylogenetic conservation of the ORC-binding site and facilitated the identification of functional ACS motifs (Theis et al 1999;Nieduszynski et al 2006;Chang et al 2008). Other studies have investigated genome replication in a wider range of Saccharomycotina species.…”