MERIT Award Recipient: John Essigmann, Ph.D.
Intra- and Extra-Chromosomal Probes for Mutagenesis by Carcinogens
This project addresses the mechanisms by which cells respond to DNA damaging agents. A specialized technology developed earlier on this project provides the ability to define the relationships between the structures of lesions formed in the genome by carcinogens and the biological endpoints of mutation, cancer and lethality. The work begins with the construction of intact viral or plasmid vectors containing, at one genome site, one of the DNA lesions hypothesized to be responsible for the mutagenesis or toxicity of a DNA damaging agent. Following introduction of the site-specifically modified genome into bacterial or mammalian cells, the genome is replicated either intra- or extra-chromosomally. We have the ability to control exposure to repair enzymes and, to a certain extent, the polymerases that encounter the lesion in vivo. The type, amount and genetic requirements for mutagenesis and lesion lethality are evaluated. By comparison of lethality and mutagenesis in genetically defined cell lines, it is possible to determine to what extent specific host factors (e.g., repair enzymes) protect, or sensitize, the cell to specific DNA or RNA lesions.
Earlier work by our laboratory focused on defining the mechanisms of action of a wide range of DNA damaging agents, including environmental carcinogens, radiation and anticancer drugs. The current project addresses the genotoxic mechanisms of three classes of agents: simple DNA alkylating agents, a furanocoumarin (aflatoxin) epoxide, and the reactive nitrogen and oxygen species associated with inflammation. By studying a carefully chosen range of DNA lesion structures, we seek to understand the strategies used in biology to address the challenge of DNA damage. While the work principally focuses on challenges to cellular DNA repair and DNA replication systems, we also shall add new dimension to our program through an attempt to understand the challenges faced by damage to RNA nucleotides. It is unclear whether the repair of RNA is a biologically significant event, and we hope that our studies with site-specifically modified RNA viral genomes will help to clarify that issue.
A final goal of the proposed work will be to construct an engineering model of the competition between DNA lesion repair and replication in vivo. DNA adducts will be placed at increasing distances from the origin of replication and the kinetics of repair will be measured at each site. Differences in lesion-induced mutation frequency from site to site, and the time it takes a polymerase to traverse the distance between the sites, will be used to compute the kinetic parameters of DNA repair enzymes in vivo.
This work impacts public health in several ways. First, our studies using cells with defects in replication and repair is helping provide a basis for defining the genes responsible for both the mutagenic and non-mutagenic processing of specific forms of DNA damage. For example, on a number of occasions we have found that the mutagenic activity of a given lesion is strikingly enhanced in a cell line unable to carry out a specific DNA repair reaction. This result identifies a likely role for that gene in protecting cells from the specific form of DNA damage being studied. Our work on AlkB is a recent example of the value of this approach. Second, our technology provides a way to view the biological fate of DNA damage in cells that are minimally, if at all, challenged by a DNA damaging agent. A common criticism of conventional toxicity bioassays is the fact that large doses of chemicals are usually required in order to produce a measurable effect in a reasonable time frame. The use of large doses compromises the validity of some studies, especially with regard to establishing relevance to expected levels of human exposure, which are usually quite low for most environmental agents. As an example, consider the situation in which a cell receives a low level insult of a chemical agent, which results in a correspondingly low level of genome modification. The cell may be unaffected by this treatment because it has a DNA repair system of finite capacity that removes this type of damage. At higher doses, however, the repair system may become saturated, and it may be that it is only at these high doses that the genotoxic effects of the chemical are realized. The advantage of using vectors containing a single adduct is that the dose of carcinogen, defined as the level of genome modification, is low, probably never exceeding a few adducts per cell. Third, there is significant translational impact to this work. Studies on mutagenesis and DNA damage obviously contribute to knowledge of the origins of genetic disease and to mechanisms of DNA damage induced cell death. The latter is of value toward understanding the mechanisms of toxicity of many DNA-acting anticancer drugs and some antiviral drugs. Working in collaboration with Lawrence Loeb and James Mullins of the University of Washington, we were able to leverage an understanding of novel mechanisms of genotoxicity to design an agent that is now in Phase IIa clinical trials. Thus, novel mechanism-based selective toxicants with therapeutic potential can be designed based upon the basic studies in this program.