This proposal is directed toward development of genetically engineered cell lines that will use reporter constructs to measure carcinogenic potential by making use of the cells' own response mechanisms to DNA damage. Our recent work with DNA polymerase delta has shown that a) it is a key DNA polymerase involved in DNA excision repair, and b) that its gene expression is induced by exposure of cultured cells to DNA damage in the form of UV irradiation. Thus, these novel findings raise a possibility that pol delta gene expression may be used as an index of DNA damage for testing the genetic toxicity of a variety of chemical and physical agents. This goal has become feasible since we have recently cloned the gene for human DNA polymerase delta and have isolated a 1.7 kb 5' upstream promoter region. The proposed studies will validate the concept that the response of the pol delta gene promoter can be used as an index of the cellular response to DNA damage, and therefore may be used as a test system to evaluate the carcinogenic potential of any given chemical or physical insults.
SPECIFIC AIM 1. The regulatory elements of the promoter region will be characterized, with specific attention to those involved in the response to cellular DNA damage. Reporter gene constructs using firefly luciferase as the reporter gene will be used to study the response of the pol delta promoter.
SPECIFIC AIM 2. The basic information obtained in Aim l will be used to modify the pol delta gene promoter to maintain those elements which are responsible for the response to DNA damaging agents. Basal response in the absence of the agents will be reduced to the minimum by mutagenesis of the promoter. The goal of this part of the work will be specifically directed to the optimization of the pol delta promoter so that it is highly sensitive and specific in its response to DNA damaging agents.
SPECIFIC AIM 3. The optimized reporter construct will be stably transfected into human cultured cell line(s). The response of the reporter gene when these cell lines are exposed to DNA damaging agents, both in the form of chemical and physical DNA damaging agents will be characterized.
| Perez, A; Leon, A; Lee, M Y (2000) Characterization of the 5'-flanking region of the gene encoding the 50 kDa subunit of human DNA polymerase delta. Biochim Biophys Acta 1493:231-6 |
| Sun, Y; Jiang, Y; Zhang, P et al. (1997) Expression and characterization of the small subunit of human DNA polymerase delta. J Biol Chem 272:13013-8 |
