Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

Abstract

ABSTRACT In the budding yeast, Saccharomyces cerevisiae , replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E ( ARS301 ) and I ( ARS302 ). Another, ARS303 , resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320 . Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302 , the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans -acting mutations that abrogate transcriptional silencing at HML ( sir3 , orc5 ), or cis -acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster ( ARS303 , ARS320 , and ARS302 ), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis - and trans -acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301 , the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML , providing additional time for HML ARSs to fire as origins.