INDUCTION OF DNA DAMAGE, MISREPAIR AND TUMOR INITIATION

University Of Nebraska Medical Center, Omaha NE

Investigators

Linked publications & trials

Abstract

The overall goal of this project is to study the mechanism of conversion of DNA damage into tumor-initiating mutations in mouse skin. Our results suggest that in pre-S phase cells, AP sites generated by these adducts in the H-ras gene, are converted into mutation by error-prone excision repair. Repair errors frequently appeared in defined sequence contexts and were detectable as mismatched heteroduplexes. The extent of repair is reduced by the presence of stable adduct in neighboring bases. We hypothesize that in addition to adduct type, the sequence context of DNA damage, DNA polymerases and the presence of neighboring DNA damage may affect the rate and fidelity of repair and thus, influence mutagenesis in the H-ras gene. In Aim 1, we propose to conduct a DB[alpha, 1] dose-response study to analyze the relationship between induction of mutations in H-ras gene and the level of depurinating adducts in SENCAR mouse skin. In Aim 2, we propose to characterize the regulation of mutagenesis in H-ras gene by stable adducts. We will treat SENCAR mouse skin with various DB[alpha,1]P metabolites (and with 7,12-dimethylbenz[a]anthracene) either singly or their mixtures to selectively change the level of total adducts or the proportion of depurinating to stable adducts. In Aim 3, using various cell-free extracts, we propose to study whether in oligonucleotides, the rate and fidelity of repair of defined AP sites varies by its proximity to putative misrepair- associated sequences and whether particular DNA polymerases could be associated with the error-prone repair. In Aim 4, oligonucleotides containing an AP sites as well as a neighboring lesion (either a stable anti-DB [alpha, 1]PDE-N6dA or estradiol-2,3-quinone-N6dA adduct, or an additional dU residue) will be synthesized. Using various cell-free repair extracts and these oligonucleotides, we propose to study the influence of neighboring lesions on the rate and fidelity of excision repair. These studies will provide a molecular understanding of the misrepair pathway that leads to tumor initiation.