The long-term goal of this project is to characterize at the molecular level a pathway of replication of UV-damaged DNA in human cells. Our hypothesis is that normal human cells contain a factor (or factors) that contributes to the replication of UV-induced pyrimidine dimers in a error-free manner and without detectable inhibition of DNA strand growth. Xeroderma pigmentosum (XP) variant cells are hypersensitive to mutagenesis and carcinogenesis by UV, even though they have normal levels of excision repair. Instead, XP variant cells have an increased sensitivity to inhibition of DNA strand growth by UV lesions present in chromosomal DNA. We have proposed to establish whether this phenomenon can be detected also during the replication of episomal DNA damaged by UV. We will determine the optimal conditions for replication in normal and XP variant cells of a plasmid carrying the SV40 genome (Specific Aim 1). We expect that both cell types will be equally proficient in replicating the undamaged plasmid. Then, we will measure the effects of a defined number of pyrimidine dimers or benzo[a]pyrene-diolepoxide-I (BPDE) adducts on the replication of the SV40 plasmid (Specific Aim 2). We expect the UV-irradiated plasmid to replicate better in normal than in XP variant cells. However, this differential response is not predicted for experiments in which the plasmid DNA will be adducted by BPDE-I. Origin-defective plasmids will be used as negative controls in our replication studies and to measure the incorporation of labeled precursors in plasmid DNA by excision repair. The data generated in this project will guide us in the development of in vitro assays that can detect differences in whole extracts of different human cells in their capabilities to carry on the replication of UV-damaged DNA templates (Specific Aim 3). Finally, we will initiate complementation studies to identify putative factors that participate in this process.
These specific aims will extend our knowledge of molecular events operating when replication forks encounter damaged sites in DNA. They seek to demonstrate the existence of carcinogen-dependent pathways of replication of damaged DNA. This project will begin to dissect into its molecular components the UV-dependent pathway. Overall, this study will focus on a DNA metabolic pathway that contributes to the protection of normal human beings from the carcinogenic effects of UV light, thus reducing their susceptibility to skin cancers.
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| Chen, Bo; Simpson, Dennis A; Zhou, Yingchun et al. (2009) Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53. Cell Cycle 8:1775-87 |
| Heffernan, Timothy P; Unsal-Kacmaz, Keziban; Heinloth, Alexandra N et al. (2007) Cdc7-Dbf4 and the human S checkpoint response to UVC. J Biol Chem 282:9458-68 |
| Lima-Bessa, Keronninn Moreno; Chigancas, Vanessa; Stary, Anne et al. (2006) Adenovirus mediated transduction of the human DNA polymerase eta cDNA. DNA Repair (Amst) 5:925-34 |
