Exposure to certain man-made and natural environmental agents poses a significant threat to human health. It is becoming increasingly clear that we need to expand our knowledge about the mechanisms by which toxic and carcinogenic environmental agents compromise human health, in order to enable us to establish rational policies to deal with this health issue. One of the major risks of exposure to harmful environmental agents is the development of cancer. Although the process of carcinogenesis is complex, it is clear that mutagenic activation of proto-oncogenes and inactivation of tumor suppressor genes plays an important role. Understanding the mechanisms by which carcinogenic agents cause mutagenesis is the long range goal of this research proposal. Many carcinogenic agents interact with DNA, causing damage to the DNA structure. If this damage is not removed by cellular DNA repair processes, the damage interferes with normal DNA replication and leads to mutations. We have been using an in vitro DNA replication assay and a simian virus 40 (SV40)-based plasmid vector (pZ189) to investigate how the replication machinery of mammalian cells responds to DNA damage and how mutations arise during replication. We have shown that UV-damaged templates can be replicated in vitro, and that this results in fixation of mutations that are similar to those that are induced by UV radiation in vivo. These results establish the utility of the in vitro DNA replication system as a tool for investigating mutagenesis on the molecular level. We now propose to reconstitute replication of damaged templates using protein fractions derived from the mammalian cell extract, to determine which components are required for the observed replication and mutation fixation. Further we propose to determine how the activities of these replication factors are influenced by the cell-wide changes that are induced when cells are treated with carcinogenic agents. In particular, we will focus on the mechanisms of the immediate inhibition of DNA replication that occurs during Gl arrest, and the apparent increase in the capacity of cells for replication and mutagenesis of damaged templates that occurs at later times following carcinogen treatment. The results of these studies should provide us with a better understanding of the process by which damage in the DNA template is converted to the sequence alterations observed in critical genes in cancer cells. Ultimately, the results of these studies may lead to the development of intervention strategies that could provide protection against the mutagenic effects of carcinogenic environmental agents.
| Yao, J; Dixon, K; Carty, M P (2001) A single (6-4) photoproduct inhibits plasmid DNA replication in xeroderma pigmentosum variant cell extracts. Environ Mol Mutagen 38:19-29 |
