RFC plays an essential role in DNA replication by loading the sliding clamp PCNA onto DNA in order to tether DNA polymerase δ to DNA. RFC consists of five subunits, one large subunit and four small subunits. The large subunit of RFC contains an extended C-terminal domain that is not present in the small subunits and whose function remains unknown. In addition to RFC, eukaryotic cells contain two more putative PCNA loaders known as RLCs. These other PCNA loaders have similar structures to RFC and contains the RFC small subunits, however the large subunit is replaced with a different protein, either Elg1 or Ctf18. The function of the three PCNA loaders is not clear. In this work the function of the Rfcl C-terminal domain (CTD) was examined. The analysis of an Rfcl CTD deletion mutant showed that the domain is essential for cell viability. <i>rfcl-44, </i>a temperature-sensitive mutant with a mutation in the C-terminal domain, displayed sensitivity to DNA damaging agents, abnormal chromosome structure and a synthetic lethal phenotype when combined with DNA replication mutants. <i>rfc5 </i>mutants were isolated as suppressors of <i>rfcl-44 </i>suggesting that the defect in <i>rfcl-44 </i>may be in the Rfcl-Rfc5 interaction. Ctf18, Dccl and Ctf8, components of Ctf18-RLC, were required for the viability of <i>rfc1-</i>44 whilst Elg1 was not. Deletion of Elg1 restored the viability of <i>rfc1-44 ctf18</i><i>Δ</i><i> </i>double mutant cells, suggesting that Elg1 plays a negative role. The negative role of Elg1 was confirmed by over-expression of Elg1 in <i>rfc1-44 </i>cells showing a lethal phenotype at permissive temperature. These results suggest that RFC plays a key role in DNA replication and that Elg1-RLC and Ctf18-RLC can play negative and positive roles respectively when RFC function is impaired.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:653407 |
| Date | January 2005 |
| Creators | Kim, Jiyoung |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12375 |
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