Regulating the origin recognition complex in yeast heterochromatin

In Saccharyomyces cerevisiae, the evolutionarily conserved Origin Recognition Complex (ORC) functions at multiple genomic positions. ORC has essential roles in both DNA replication initiation and the assembly of transcriptionally repressive chromatin domains (silencing). We hypothesize that characteristics of specific genomic positions may influence ORC's activity within those regions. This dissertation focuses on how specific regions within the cryptic mating-type locus HMRa can modulate ORC's replication and silencing functions at this locus.; Initial studies indicated that differences in ORC binding sites within HMRa could profoundly influence ORC function. Specifically, when natural HMR-E was the only silencer controlling HMRa, the silencer's ORC binding site (ACS or A-element) was not required for replication initiation but was essential for silencing. The regions flanking HMR-E could provide origin activity at HMRa but could not function in silencing despite being within HMRa. Unlike replication initiation by the intact silencer, replication initiation by the non-silencer origins was reduced in an orc2-1 mutant. Unexpectedly, reduced silencing correlated with the activity of the non-silencer origin at HMRa; specifically, silencing by a compromised silencer (HMRa (A-)) was enhanced by removal of the non-silencer origin activity by either the orc2-1 mutation or by deletion of the relevant DNA sequences. These data suggest that ORC bound to the silencer suppresses the function of nearby regions capable of antagonizing silencing at HMRa.; Additional studies characterized ORC/DNA interactions that promoted replication and silencing. In vitro assays indicated that ORC bound with a higher affinity to HMR-E compared to several replication origins. Furthermore, high affinity binding suppressed replication initiation within and delayed replication time at HMRa. These data are consistent with tight binding of ORC to its site within an origin leading to suppressed initiation and suggest that ORC disengages from double-stranded origin DNA to ensure efficient replication initiation. Moreover, a high affinity ORC/DNA interaction at HMR-E promoted silencing; implying that tight binding of ORC to its site within the HMR-E silencer contributes to the stabilization of silent chromatin. Therefore, high affinity interactions between silencing components may contribute to heterochromatin formation, and a high affinity ORC-chromatin interaction may be a defining feature of heterochromatic origins.