In Saccharomyces cerevisiae, DNA replication initiates at distinct origins termed Autonomously Replicating Sequences (ARSs). A key element in ARS is the ACS/B1 sequence, which binds the Origin Recognition Complex (ORC). During early G1 phase, the pre-replication complex is assembled by ORC. These ARSs are termed replicators. In yeast, ORC is also involved in gene silencing. These loci also contain an ACS/B1 element; however at these positions ORC recruits the silencing SIR complex. ARSs found here are termed silencers. Therefore, ARSs have a dual function. Research has also shown that origin ARSs can be substituted for silencing ARSs and vice versa. Since a mutation in ACS abolishes replication activity, studies have turned to the B1 element to account for the functional duality, but results have been mixed. I hypothesize that the B1 element plays a key role in the dual nature of ARS. To test this hypothesis, silencer and replicator ARSs were subjected to site directed mutagenesis around the WTW motif of the B1 element. Their efficiency was then tested using routine silencing and replication assays. Results reveal that the silencing ability of silencer ARSs is unaffected by these mutations; however mutations within and around the WTW motif reduce silencing efficiency of replicator ARSs. Sequence alignments have also shown that silencer ARSs have a broader WTW consensus than replicator ARSs. Preliminary replication assays are consistent with the above results and other research, contributing to a conclusion that the B1 element is not the sole determining factor in the dual nature of ARS. / NSERC
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/3331 |
| Date | 16 February 2012 |
| Creators | Chisamore-Robert, Patricia |
| Contributors | Yankulov, Krassimir |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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