Double-strand breaks (DSBs) are one of the most lethal threats to genomic integrity. In the yeast Saccharomyces cerevisiae, double-strand breaks are primarily repaired via the homologous recombination repair pathway. While the mechanisms underlying this pathway have been generally characterized, the early steps leading to commitment of DSBs to homologous recombination repair are as yet unclear. In particular, it has been shown that the exonuclease processing of DSBs is a critical step prior to strand invasion of a homologous template. / Endo-exonucleases represent a class of nucleases that have been linked to recombinational repair processes in various organisms. Early on, a degradative role was ascribed to the bacterial recBCD endo-exonuclease in processing DSBs in a 5' to 3' directionality prior to recA-mediated homologous recombination in E. coli. However, the nuclease responsible for this nucleolytic processing has yet to be identified in yeast. In this dissertation, I demonstrate that RNC1 is a candidate gene that encodes a nuclease with characteristics similar to previously identified endo-exonucleases, and that RNC1 is implicated in homologous recombination repair of DSBs. / While the rnc1 null mutant is 10-fold more sensitive to persistent DSB induction than wild-type, a highly lethal rad52 null mutation is suppressed in an rnc1 knockout, indicating that RNC1 acts at an earlier step in the homologous recombination repair pathway than RAD52. Furthermore, in the absence of RNC1, the non-homologous end-joining pathway, mediated by the KU80 gene product, appears to play a more dominant role in repairing DSBs. Moreover, a ku80 rnc1 double mutant is hypersensitized to DSB induction. / Biochemical analysis of the purified RNC1 protein revealed that it has endonuclease activity on single-strand DNA as well as processive exonuclease activity on double-strand DNA with a 5' to 3' directionality. In vivo, this exonuclease activity was implicated in the processing of DSBs prior to repair. Our findings show that RNC1 possesses nucleolytic activities characteristic of previously identified endo-exonucleases, and confirm its role in the homologous recombination repair pathway. Intriguingly, RNC1 was also shown to exhibit methyltransferase activity which directs the modification of a specific base, m5U 54, in elongator tRNA. While the biological significance of this activity is as yet unclear, it is attributable to a domain in the RNC1 protein separate from that engaging in DNA repair processes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37644 |
| Date | January 2001 |
| Creators | Asefa, Benyam. |
| Contributors | Chow, Terry (advisor) |
| Publisher | McGill University |
| 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 | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001864245, proquestno: NQ78641, Theses scanned by UMI/ProQuest. |
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