Using Nucleotide Analogs as Biochemical Probes to Evaluate the Mechanisms Involved in Translesion Replication by a High Fidelity DNA Polymerase

Translesion DNA synthesis (TLS) allows DNA polymerases to incorporate nucleotides opposite and beyond damaged DNA. This activity is an important risk factor for the initiation and progression of genetic diseases including cancer. My study evaluates the ability of a high-fidelity DNA polymerase to perform TLS with 8-oxo-guanine, a pro-mutagenic DNA lesion formed by reactive oxygen species. Using modified purine and non-natural indole analogs as biochemical probes, I have evaluated the influence of desolvation, hydrogen bonding interactions, and shape complementarity towards nucleotide binding and incorporation opposite the miscoding lesion 8-oxo-guanine by the high fidelity gp43exo- DNA polymerase. In Chapter II, I used modified purine nucleotide analogs to provide evidence that nucleobase desolvation and hydrogen bonding interactions play a crucial role towards binding and incorporation opposite 8-oxo-guanine. This was further confirmed by studies in Chapter III through kinetic characterization using non-natural indole nucleotide analogs. Overall, I have demonstrated that the binding affinity of the incoming dNTP is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for the conformational change preceding chemistry is regulated by hydrogen-bonding interactions and play a much larger role during the replication of miscoding lesions such as 8-oxo-G. Results generated here for the replication of 8-oxo-guanine were compared to those published for the replication of an abasic site, a non-instructional DNA lesion. With both lesions, nucleobase hydrophobicity is a common feature that controls nucleotide binding whereas the physical nature of the lesion, i.e., miscoding versus non-instructional, influences the rate constant of the conformational change step that precedes phosphoryl transfer. Collectively, these studies highlight the importance of nucleobase desolvation as a key physical feature that can hinder or facilitate the misreplication of structurally diverse DNA lesions. In Chapter IV, I have investigated the unique selectivity of a modified purine analog to DNA polymerases with varied biological function. Active analysis demonstrated that differential architecture of high and low fidelity DNA polymerases plays a predominant role in the replication of damaged DNA. Therefore, this differential utilization provides a unique opportunity to develop a chemical probe to monitor translesion DNA synthesis in-vivo