Replicative Bypass of a DNA-peptide Cross-link and Oxidative Single and Tandem DNA lesions in Human cells

DNA harbors the genetic information in all living organisms and plays a pivotal role in passing the genetic information to the next generation. All living cells are exposed to numerous exogenous and endogenous agents that damage the DNA to form different lesions or adducts in DNA. Fortuitously, diverse DNA repair mechanisms exist in the living cells, which repair these DNA adducts before the replication apparatus of the cell copies the DNA. However, frequently extensive DNA damage or slow repair may allow replication bypass of a DNA lesion. As a result, cells adapt a DNA damage tolerance mechanism, termed as translesion synthesis (TLS) to bypass the lesions in DNA. Often, the TLS polymerases are of low fidelity and result in the misincorporation of the nucleobases generating mutations. If these mutations persist in the DNA, it may lead to alteration of genetic code and protein expression, thereby resulting in various diseases including cancer. In this thesis, I investigated the mutagenicity and genotoxicity of three different adducts including a bulky DNA-peptide cross-link, a tandem lesion containing 2-deoxyribonolactone with thymine glycol and oxidative stress induced formamidopyrimidine dG (Fapy.dG) in human embryonic kidney (HEK293T) cells. My goal is to investigate the role of each of the TLS polymerases (pol 𝝶, pol 𝝹, pol 𝝸, pol 𝝵), by replicating these lesions in selective polymerase-deficient human cell lines and determine the types and frequencies of mutations. From these analyses, it was discovered that these adducts are bypassed by TLS mechanism in both error-prone and error-free manner by the action of the specific TLS polymerases. From these studies, it was evident that the replicative bypass depends of the structure, DNA sequence context, and the orientation of the lesion in DNA. This research provides an insight to better understand the mutagenicity and toxicity of these lesions in human cells, which should benefit gene therapy applications in the future.