Expanded Roles of the Origin Recognition Complex in the Architecture and Function of Silenced Chromatin in Saccharomyces cerevisiae

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2009; 30(3):626-39. DOI: 10.1128/MCB.00614-09
Source: PubMed


The silenced chromatin at the cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae requires a cell cycle event between early S phase and G2/M phase to achieve repression. Although DNA replication per se is not essential for silencing, mutations in many of the proteins involved in DNA replication affect silencing. Each of the
four silencers, which flank the silenced loci, includes an origin recognition complex (ORC) binding site (ACS). ORC directly interacted with Sir1 and recruits Sir1 to the silencers. This study describes additional roles for ORC in
the architecture of silenced chromatin. Using chromatin immunoprecipitation (ChIP) analysis, we found that ORC physically
interacts throughout the internal regions of HMR as well as with silencers. This interaction depended on the presence of Sir proteins and, in part, on the HMR-I silencer. ORC remained associated with the internal regions of HMR even when these regions were recombinationally separated from the silencers. Moreover, ORC could be recruited to the silencers
lacking an ACS through its Sir1 interaction.

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Available from: Bilge Ozaydin, Oct 31, 2014
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    • "Thus, Sir1 and ORC serve to nucleate the remaining Sir proteins which spread across the locus, resulting in hypoacetylation and structural changes in the chromatin leading to the loss of transcription. Recent high-resolution ChIP studies from the Rine laboratory suggest that ORC may have a more extensive role in silencing and perhaps contribute to the local chromatin structure and organization of silenced sequences (Özaydin and Rine, 2010). "
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    ABSTRACT: DNA replication is an essential cell cycle event required for the accurate and timely duplication of the chromosomes. It is essential that the genome is replicated accurately and completely within the confines of S-phase. Failure to completely copy the genome has the potential to result in catastrophic genomic instability. Replication initiates in a coordinated manner from multiple locations, termed origins of replication, distributed across each of the chromosomes. The selection of these origins of replication is a dynamic process responding to both developmental and tissue-specific signals. In this review, we explore the role of the local chromatin environment in regulating the DNA replication program at the level of origin selection and activation. Finally, there is increasing molecular evidence that the DNA replication program itself affects the chromatin landscape, suggesting that DNA replication is critical for both genetic and epigenetic inheritance.
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    • "Alternatively, in light of a recent study showing that ORC binding spread throughout the HMR silent domain rather than being restricted to the HMR silencers (52), it is also possible that ORC similarly binds HML beyond the silencer, and that this binding, and thus the contribution of ORC to silencing, is not abrogated by orc2-1 and orc5-1. "
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    ABSTRACT: The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ΔI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.
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    • "Transformants were selected by auxotrophic complementation and the functionality of the recombinant strains analyzed for testing their ability to produce CO2 in high-sucrose LD. However, only overexpression of CAF16 and ORC2, two of the six transcriptional factor-encoding genes [14-16], identified as specifically induced in high-sucrose LD (additional file 2), had significant positive effects on leavening activity of baker's yeast cells (Table 3 and Figure 3, control, 0 days). Because of this, only strains overexpressing these two genes were further characterized. "
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