Identification of a Compound Origin of Replication at theHMR-E Locus in Saccharomyces cerevisiae

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.The replication of eukaryotic chromosomes initiates at multiple origins during each S phase. These DNA replication origins are best understood in the budding yeast Saccharomyces cerevisiae, in which they were initially recognized by their ability to promote the autonomous replication of plasmids. For this reason, they are referred to as autonomously replicating sequence (ARS) elements (29, 55). The paradigm S. cerevisiae replication origin is ARS1. It has a modular structure that spans about 120 bp and includes a small essential region, domain A, and three small accessory sequences, B1, B2, and B3, mutations in which reduce but do not abolish activity (40). Domain A, which encompasses the essential match to the 11-bp ARS consensus sequence (ACS), is the core of the binding site for the S. cerevisiae replication initiator protein, the origin recognition complex (ORC). The six-subunit ORC complex also contacts and protects DNA in the B1 element, and some mutations in B1 compromise ORC binding in vitro (3, 37, 50, 52). The B3 element contains a binding site for the transcriptional activator-repressor Abf1p, which can be replaced by the binding sites for the transcriptional regulators Rap1p and Gal4p (40). The precise role of the B2 element has not been defined, although at least one of its functions is likely to be unwinding the DNA duplex to allow entry of the replication machinery (38, 41).Other well-studied ARS elements, including ARS307 (49, 57), ARS305 (30), ARS121 (62), and the H4 ARS (7), seem to fit the ARS1 paradigm in that they contain a single, essential ACS flanked by a B domain. However, details of the structure of the B domain differ, and some ARS elements also contain stimulatory sequences on the other side of domain A, a region called domain C.In this paper, we describe our characterization of two ARS elements, ARS101 and ARS310,...

Section: Discussionmentioning

confidence: 99%

Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

Theis

Newlon

2001

“…However, mutation of the ACS abolishes ARS activity only within a minimal 138-bp fragment containing the silencer elements, i.e., the ORC, Rap1p, and Abf1p binding sites (Fig. 5B) (19,28,33). In other words, if natural sequences flanking this 138-bp minimal fragment are present, the ACS Ϫ mutant retains replicator activity both in the plasmid and on the chromosome.…”

Section: Vol 28 2008mentioning

confidence: 99%

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

Chang

Theis

Miller

et al. 2008

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...

“…The HMR-E silencer is depicted by the black box. The gray boxes surrounding the silencer represent regions that contain near matches to the A-element and contribute to ORC binding and origin firing in HMR-E(A-) (18,26). Natural HMR-E consists of binding sites for ORC (B1/A-element [black box]) and Rap1p and Abf1p (white boxes).…”

Section: Resultsmentioning

confidence: 99%

“…The experiments described above using HMR-E(A-) mutant cells indicated that Sir1p-dependent silencing could tolerate enhanced origin firing, or something about ORC that promoted this origin firing, at HMRa. However, since multiple regions within HMRa can bind ORC in vitro (M. A. Palacios DeBeer, unpublished) and function as origins in vivo (18,26), these experiments could not address whether the same ORC molecule that bound Sir1p could also function in enhanced origin firing. To better address this issue, we used an engineered version of the HMR-E silencer named the synthetic silencer (HMR-SS) that contains a single ORC binding site required for both origin firing and silencing at HMRa (23,32) (Fig.…”

Section: Sir1-dependent Silencing Of Hmr-e(a-) [Sir1 (ϩϩϩ)] Did Not Dmentioning

confidence: 95%

“…This apparently paradoxical result, given ORC's established positive role in origin firing, can be explained by the presence of several neighboring near matches to the A element surrounding the defined HMR-E silencer (21) that can bind ORC in vitro (M. A. Palacios DeBeer and C. A. Fox, unpublished) and provide for ORC-dependent origin firing in vivo (18,26). We postulate that when an ORC normally binds its site within HMR-E, it somehow prevents additional ORCs from binding to the several near matches to the A element surrounding the silencer.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing atHMRa

McConnell

Müller

Fox

2006

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.