Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

Abstract

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.The complex process of chromosomal DNA replication in eukaryotes is poorly understood, partly because origins of DNA replication are not well characterized. In Saccharomyces cerevisiae, autonomously replicating sequences (ARS elements) promote efficient plasmid replication and thus permit plasmids containing them to transform yeast cells at high frequency without integration into the genome. ARS elements can be classified as strong or weak, depending on the mitotic stabilities of plasmids containing them. Strong ARS elements promote high mitotic stabilities, and weak ARS elements promote lower mitotic stabilities. The properties of ARS elements suggest that they may serve as replication origins (7,47 see references 40 and 40a for the locations of these ARS elements on chromosome III)-and a weak ARS in tandemly repeated ribosomal DNA (31) are all active as chromosomal replication origins. In ribosomal DNA, however, only 5 to 30% of available ARS elements are active as origins in each cell cycle (31). Whether all the ARS elements in nonrepeated chromosomal DNA are active as replication origins was, until recently, unknown. The experiments described in this paper reveal that some ARS elements in unique DNA do not detectably function as origins in their normal chromosomal environment.In S. cerevisiae, there are two mating types, a and a. Information specifying mating types is present at three * Corresponding author. t Present address: Department of Zoology, Kutir Mahavidyalaya, Chakkey, Jaunpur-222146, U.P., India.locations on chromosome III, the HML, MAT, and HMR loci (Fig. 1). Under normal conditions, only the information at the MAT locus (either a or a) is expressed. Haploid yeast cells can switch mating type by replacing the MAT information with a copy of either the information present at HML (usually a) or the information present at HMR (usually a) (reviewed by Herskowitz [22]). The HML and HMR loci are located near the left and right telomeres of chromosome III, and infor...

Replication profile of Saccharomyces cerevisiae chromosome VI

“…Surprisingly, the level of origin activity is extremely variable from origin to origin, with about half of the origins activated in 50% or fewer cell cycles. Although inefficient or inactive origins had previously been found on chromosome III, these inefficiencies seemed likely to result from the association of the ARSs with the silent mating type loci and with other peculiarities of chromosome III related to mating-type switching (Dubey et al 1991). Furthermore, the few origins randomly identified on other chromosomes had generally been found to be very efficient (e.g.…”

“…Some, but not all of these ARSs have been shown to function as chromosomal origins of replication through analysis of replication intermediates by two-dimensional (2D) gel electrophoresis (e.g. Huberman et al 1988;Dubey et al 1991;Ferguson et al 1991;reviewed in Newlon & Theis 1993). Restriction fragments containing yeast replication origins have subsequently been shown to replicate at very different times during S phase.…”

“…Interestingly, most inactive origins on the chromosome are contained within or proximal to sequences that maintain a silent chromatin context for the genes involved in the gene conversion event that switches yeast mating type (HML and MAT, Dubey et al 1991). Thus, the fact that over half of the potential origins in this segment of chromosome III are inactive may be related to unusual chromatin context associated with the silent mating type loci, though transcriptional silencing itself is not sufficient to repress origin activation (Dubey et al 1991). Timing experiments suggest that origins located near the middle of chromosome III are activated quite early in S phase, and that the left telomere of the chromosome is late replicating due to passive replication by slow-moving forks initiated at distal, early origins (Reynolds et al 1989).…”

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

“…The establishment of silent chromatin requires a passage through S-phase (Miller and Nasmyth 1984), but the requirement for DNA replication per se has been ruled out (Dubey et al 1991;Kirchmaier and Rine 2001;Li et al 2001;Sharma et al 2001). Nevertheless, the list of DNA replication factors that affect silencing has been steadily growing (EhrenhoferMurray et al 1995(EhrenhoferMurray et al , 1999Dillin and Rine 1997;Fox et al 1997;Dziak et al 2003;Suter et al 2004;Liachko and Tye 2005).…”

“…The establishment of silent chromatin requires passage through S-phase (Miller and Nasmyth 1984), but does not require DNA replication (Dubey et al 1991;Kirchmaier and Rine 2001;Li et al 2001). Furthermore, robust silencing at HMR does not take place until M-phase (Lau et al 2002).…”