Mutagenic Potential of Alkyl-substituted and unsubstituted Fapy-dG 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. 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 κ 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.
- Harbut MB, Meador M, Dodson ML, Lloyd RS. Modulation of the turnover of formamidopyrimidine DNA glycosylase. Biochemistry. 45(23):7341-6, 2006. PMID: 16752923
- Minko, I.G., Kozekov, I.D., Harris, T.M., Rizzo, C.J., Lloyd, R.S., Stone, M.P. Chemistry and biology of DNA containing 1,N(2)-deoxyguanosine adducts of the alpha, beta-unsaturated aldehydes acrolein, crotonaldeyde, and 4-hydroxynoneal. Chem Res Toxicol 22(5):759-78, 2009. PMID: 19397281, PMCID: 2685875.
- Christov PP, Yamanaka K, Choi JY, Takata KI, Wood RD, Guengerich FP, Lloyd RS, Rizzo CJ. Replication of the 2,6-Diamino-4-hydroxy-N(5)-(methyl)-formamidopyrimidine (MeFapy-dGuo) Adduct by Eukaryotic DNA Polymerases. Chem Res Toxicol. 25(8), 1652-1661, 2012 PMID: 22721435 PMCID: 3682502
- Yamanaka K, Dorjsuren D, Eoff RL, Maloney DJ, Jadhav A, Simeonov A, and Lloyd RS. A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ. PLoS ONE 7(10): e45032, 2012 PMID: 23056190 PMCID:3147505
- Chary, P, Beard, WA, Wilson, SH, Lloyd, RS. DNA polymerase β gap-filling translesion DNA synthesis. Chem Res Toxicol. 25(12): 2744-2754, 2012 PMID: 23121263 PMCID: 3523550
- Earley, LF, Minko, IG, Christov, PP, Rizzo, CJ, Lloyd RS. Mutagenic spectra arising from replication bypass of the 2,6-diamino-4-hydroxy-N5-methyl formamidopyrimidine adduct in primate cells. Chem Res Toxicol 2013 PMID: 23763662 PMCID: not assigned
- Lin, YC, Makarova, AV, Burgers, PM, Stone, MP, Lloyd, RS. Molecular basis of aflatoxin-induced mutagenesis – role of the aflatoxin B1 formamidopyrimidine adduct. 2014 Carcinogenesis e-pub
- Lin, YC, Makarova, AV, Burgers, PM, Stone, MP, Lloyd, RS. Error-prone Replication Bypass of the Primary Aflatoxin B1 DNA Adduct, AFB1-N7-Gua 2014 J. Biol. Chem. E-pub
- Minko IG, Earley LF, Larlee KE, Lin YC, Lloyd RS. Pyrosequencing: applicability for studying DNA damage-induced mutagenesis. Environ Mol