The characterization of rodent cell mutants hypersensitive to UV light has led to the identification of at least 10 complementation groups all defective in some aspect of the first step in the excision repair of UV damaged DNA. The phenotypic properties of these mutants are thus of considerable importance to our understanding of DNA repair. In recent years five different excision repair cross complementing (ERCC) human genes have been isolated which correct the DNA repair deficiency in a number of Chinese Hamster Ovary (CHO) cell mutants and at least three of these genes also complement the repair deficiency in cells from patients suffering from xeroderma pigmentosum (XP), Cockayne syndrome (CS) and/or Trichothiodystrophy (TTD). Adenovirus (Ad) infection of rodent cells is generally semi-permissive and does not give rise to viral progeny, such that Ad reactivation in CHO cells has not previously been reported. This study utilizes the ability of CHO cells and human cells to replicate viral DNA in order to examine the reactivation of Ad in several CHO as well as human cell DNA repair mutants. Unirradiated and UV-irradiated suspensions of Ad were assayed for their ability to synthesize viral DNA following the infection of several CHO and human cell DNA repair mutants. The cell types examined included CHO cell mutants from complementation groups 1, 2, 3, 4, 5, 6, 9, 10 as well as human XP and tumour cells. The survival of viral DNA synthesis for UV-irradiated Ad was significantly reduced in several of the CHO and human cell mutants compared to that in normal cells. Cell mutants showing a reduced UV survival for this viral function included CHO cell mutants from complementation groups 1 to 6, XP cells and the 2 human tumour cell lines examined. This reduced host cell reactivation (HCR) for Ad indicates a reduced capacity for the repair of viral DNA in these cell types. DNA replication for unirradiated virus was also reduced for some of the mutants, especially the UV20 CHO cell mutant from complementation group 1, suggesting a deficiency for both DNA replication and repair in these cells. This study also used the recombinant viruses Ad5(denV) and Ad5(ERCC1) as vectors to examine the effect of the bacteriophage T 4 denV gene and the human ERCC1 gene on viral reactivation in the various cell mutants. UV survival of Ad5(denV) was increased compared to that of the control Ad5(LacZ) following infection of all the CHO and human cell types examined, indicating the denV gene product increases repair of Ad in both repair-proficient and repair-deficient cells. UV survival of Ad5(ERCC1) was increased compared to that of control Ad5(LacZ) following infection of the CHO mutant UV20 from complementation group 1, as well as all CHO cell types having normal HCR for AdS. However, UV survival of Ad5(ERCC1) was not increased compared to Ad5(LacZ) following infection of CHO mutants from complementation groups 2 to 6 and 10. These results support a specific complementation of the UV20 repair defect by ERCC1 and suggest that the human ERCC1 gene is more efficient than its hamster counterpart in repair-proficient CHO cells or that the ERCC 1 product is rate-limiting for the excision repair process in CHO cells. UV survival of Ad5(ERCC1) was also increased compared to Ad5(LacZ) in the normal human fibroblast cells and human tumour cells, but not in the XP (group D) cells. The kinetics of viral DNA synthesis and viral protein synthesis for unirradiated Ad5(denV) and Ad5(ERCC1) was also investigated following the infection of human and rodent cells. The deficiency in viral DNA synthesis and viral protein synthesis found for AdS(LacZ) following infection of rodent compared to human cells is partially complemented by either denV or ERCC1. The more marked deficiency in viral DNA synthesis of the UV20 CHO mutant was also complemented by either ERCC1 or denV, suggesting an ability of these genes to function in both repair and replication of viral DNA. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23142 |
| Date | 08 1900 |
| Creators | Arnold, Wayne |
| Contributors | Rainbow, Andrew, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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