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 if the development of cancer. Although the process of carcinogenesis is complex, it is clear that mutagenic activation of proto-oncogenes 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. It this damage is not removed by cellular DNA repair processes, the damage interferes with normal DNA replication and causes mutations. The molecular mechanisms by which mutation fixation occurs are not well understood. We propose to use mammalian in vitro DNA replication systems and a simian virus 40 (SV 40)-based plasmid vector (pZ189) to investigate how the replication machinery of the cell responds to DNA damage and how mutations arise during DNA replication. SV 40 viral DNA replication has long been used as a model for mammalian chromosomal DNA replication. Now the in vivo SV 40 replication process can be duplicated in vitro. In addition, SV 40- based shuttle vector plasmids that have been used for mutation analysis in vivo, can now be used for analysis of mutations that occur in vitro. The response of the cellular replication machinery to damage in the template DNA, will be assessed by analyzing the synthesis and structural characteristics of DNA replication intermediates produced when ultraviolet (UV)-irradiated or benzo[a]pyrene diol-epoxide (BPDE)-treated pZ189 plasmid DNA is replicated in vitro. It will be of particular interest to determine whether any damaged molecules can become completely replicated, as they do in vivo, since it is during this process of damage accommodation that mutagenesis is thought to occur. To investigate mutation fixation in vitro, damaged pZ189 that has been replicated in vitro will be screened (with an E. coli testor strain) for mutations in the supF mutagenesis target gene of the vector. The supF genes of these mutants will be sequenced to determine whether the spectra of mutations induced in vitro resemble those observed in vivo. The components of the DNA replication system that are required for damage accommodation and for mutagenesis will be sought by using partially fractionated in vitro systems. The results of these studies should establish the in vitro replication system as a powerful tool for addressing questions relating to the toxic and mutagenic effects of carcinogenic agents and provide us with a better understanding of molecular mechanisms of mutagenesis.
| 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 |
