Sharon King Bullock, Ph.D.

Dissertation research performed under the guidence of Dr. Marila Cordeiro-Stone

ABSTRACT
    In this research project, we analyzed the response to UV of fibroblasts from normal individuals and from patients with the autosomal recessive disease xeroderma pigmentosum variant (XPV). XPV cells are proficient in DNA nucleotide excision repair (NER) of pyrimidine dimers, yet defective in post-replication repair (PRR). Historically, the inhibition of DNA replication in human cells has been thought to result exclusively from the blockage of the replication machinery by the UV-induced photoproducts (cis-acting effects of, the lesions). We hypothesized that UV-induced blockage of DNA replication and production of daughter-strand gaps also induces trans-acting factors that inhibit the displacement of other replication forks before they reach any damage. Because XPV cells are defective in bypass replication of pyrimidine dimers, blocking of replication and production of daughter-strand gaps are enhanced in UV irradiated XPV cells, as compared to normal controls. We predicted therefore, that a signal transduction pathway leading to inhibition of DNA synthesis in undamaged replicons would also be enhanced in XPV cells. We first transformed secondary cultures of XPV fibroblasts with SV40 large T antigen (King et al., Exp. Cell. Res., 217, 100-108, 1995) and confirmed that the XPV cell lines displayed levels of UV-induced inhibition of DNA replication comparable to their diploid counterparts. Then we characterized the UV dose-dependent inhibition of replication of a SV40-based episomal DNA (pSV011) that was transfected into transformed cell lines from PRR-normal (IDH4) and XPV (CTag) cells. By assaying for the extent of replication of pSVO11 when the host cell was irradiated (1-4 J/m²) 15 h after transfection, we showed that the IDH4 cells were barely affected, however the XPV cells displayed about a 50% inhibition of pSVO11 replication. Experiments evaluating UV cytotoxicity confirmed that this response by CTag cells could not be accounted for by cell killing. In addition flow cytometric analyses done to measure the effects of radiation (UV and gamma) on cell cycle progression showed that CTag displayed a more enhanced S phase arrest than IDH4 cells, yet neither cell type expressed S phase checkpoint responses to irradiation (gamma or UV). These results taken together suggested that the reduced recovery of pSVO11 replication products from CTag could be the result of an activation of a S phase arrest response to UV irradiation. We believe that the UV-induced inhibition of pSVO11 replication observed with CTag is the result of the activation of a S phase arrest response, independent of inhibition of replicon initiation. Based on this premise, UV irradiated IDH4 cells did not display measurable inhibition of pSVO11 replication because in these cells low doses of UV did not elicit the activation of the S phase checkpoint.