DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is a relatively fast and error-free repair pathway for damaged DNA, which can occur through a gene conversion event or through a crossing-over event with the exchange of genetic material. Homologous recombination occurs most frequently in the G2 phase of the cell cycle and utilizes the sister chromatid as the repair template. When the sister chromatid is unavailable, the homologous chromosome or a homologous sequence in an ectopic location can be used to repair the lesion; the latter of which is referred to as ectopic homologous recombination (EHR). Rad51 is a key protein involved in HR, and to test its role in EHR, variant Rad51 proteins were expressed in murine hybridoma cells. These Rad51 variants were assayed for their effects on EHR. Excess wild-type Rad51 as well as a deficiency of wild-type Rad51 decreased EHR from the background level found in these cell lines. Thus, Rad51 is necessary for EHR, but there may be an optimal amount of Rad51 required for efficient EHR. Expression of the Rad51 catalytic mutants Rad51K133A and Rad51K133R was found to have an inhibitory effect on EHR, as expected based on the loss of ATP binding and ATP hydrolysis, respectively, in these variants. Excess wild-type Rad51 was verified in this study to increase HR via a gene targeting assay. MMC treatment, but not ionizing radiation, leads to an increase in EHR in the presence of excess wild-type Rad51. Thus, endogenous levels of Rad51 are sufficient to maintain EHR, but in the presence of excess wild-type Rad51, the level of EHR can increase in response to certain DNA damaging agents and in response to gene targeting.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/7280 |
| Date | 12 July 2013 |
| Creators | Knapp, Jennifer |
| Contributors | Baker, Mark |
| 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 |
| Rights | Attribution-NonCommercial-NoDerivs 2.5 Canada, http://creativecommons.org/licenses/by-nc-nd/2.5/ca/ |
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