Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells

Abstract

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O2-methylthymidine (O2-Me-dT) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT). To investigate the biological effects of these O2-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O2-Me-dT or O2-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O2-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O2-Me-dT was less genotoxic and allowed 55% TLS. However, O2-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O2-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T→A transversions (56% and 47%, respectively, for O2-Me-dT and O2-POB-dT). Both lesions induced a much lower frequency of T→G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O2-Me-dT and O2-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O2-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O2-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.