Context-dependent modulation of replication activity of Saccharomyces cerevisiae autonomously replicating sequences...

Evidence for transcription factor involvement in the initiation of DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere. In order to eliminate the effect of transcription factor binding to the selectable marker gene or centromere in such assays, we have adapted the DpnI assay to directly measure ARS replication activity in vivo by using ARS plasmids devoid of extraneous transcription elements. Using this assay, we found that the B3 element of ARS1, which serves as a binding site for the transcription factor Abf1p, does not stimulate ARS activity on plasmids lacking a centromere and a selectable marker gene. We also found with such plasmids that exogenous expression of the strong transcriptional activators Gal4 and Gal4-VP16 inhibited the replication activity of ARS1 when B3 was replaced by the Gal4 binding site, although these activators had previously been shown to stimulate replication activity in the stability assay. Moreover, a chromosomally inactive ARS, ARS301, which was active by itself on a plasmid, was inactivated by placing an Abf1p binding site in its vicinity. These results indicate that the sequences surrounding the ARS as well as properties of the ARS element itself determine its response to transcription factors.