Mutagenic Potential of Alkyl-substituted and unsubstituted Fapy Adducts
Studies are designed to investigate the mechanisms that modulate the mutagenic potential of unsubstituted and a series of N5-alkyl-substituted Fapy-dG DNA adducts by determining the frequency and spectra of mutations formed following replication in wild-type primate cells. It is hypothesized that both the mutation frequency and spectra will be significantly modulated by 1) the local sequence context of the adduct, 2) the steady-state stereochemical equilibrium of the adduct at the replication fork, and 3) the size of the N5-Fapy-dG adduct.
Most Recent Work
The specific DNA adducts that will be investigated represent a continuum of sizes and structural complexities. Investigations will be carried out in collaboration with Dr. Carmelo Rizzo at Vanderbilt University, to test replication models consistent with the generation of transversion and/or deletion mutations. These data will serve as a guide for biophysical studies in collaboration with Dr. Michael Stone at Vanderbilt University that will discern the structural basis for N5-alkyl-Fapy-dG-induced mutagenesis.
Further objectives are to identify DNA polymerases within mammalian cells that are responsible for low and high fidelity bypass of the unsubstituted and alkyl-Fapy-dG adducts and to determine the DNA repair systems that respond to these lesions. Based on preliminary data correlating in vitro replication bypass studies with observed mutations generated in primate cells, it is hypothesized that pol k and possibly pol η are primarily responsible for TLS of these adducts. To test this hypothesis, we propose the following strategies: 1) siRNA depletion of individual polymerases and repair enzymes to modulate the absolute levels of these enzymes within COS-7 cells, during the time of replication of the shuttle vector, 2) addition of small molecule chemical inhibitors of specific TLS DNA polymerases and DNA glycosylases during replication bypass, and 3) replication of ds DNA vectors containing site-specific adducts through MEFs derived from control and polymerase-deficient mice.
This is a new project, so check back soon for updates on our progress!