Date of Award
Doctor of Philosophy (PhD)
Polymerases are vital enzymes in the continuation of life, responsible for the replication of genetic material and the conversion of genetic information to necessary products. A large subset of these polymerases is dedicated to the high-fidelity replication and repair of DNA in the cell cycle of organisms. In addition, viruses utilize polymerases in order to produce DNA or RNA used to synthesize products for virion assembly. With such an important role, polymerases have been a focus in many therapeutic studies of cancer and antiviral treatments. This dissertation focuses on three different polymerases, PrimPol, human immunodeficiency virus (HIV) reverse transcriptase (RT), and DNA polymerase α (Polα). The goal of this work was to understand their overall mechanisms and roles not only in the context of replication and repair, but also in antiviral therapies. HIV treatment, typically referred to as highly active antiretroviral therapy (HAART), consists of drugs that target various enzymes important for viral life cycle. A major fraction of these compounds, which target RT, can be classified into nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). One prevailing issue with NRTIs is that administration of these drugs may cause off-target toxicity within patients, affecting adherence to treatment regimens. This off-target toxicity can be attributed to the incorporation of NRTIs by host polymerases, such as the mitochondrial polymerase γ (Polγ). To this end, I investigated the possibility of PrimPol, a recently characterized polymerase, in mediating the mitochondrial toxicity effects seen in HIV+ patients taking tenofovir (TFV)-containing treatments. Using gel-based kinetic assays, I validated that the active metabolite form of tenofovir is a substrate for PrimPol. Cellular-based assays using overexpression and knockdown PrimPol renal cells suggests that PrimPol likely plays a protective role against tenofovir-induced toxicity through its repriming activity, despite the in vitro incorporation evidence. Given this potential role of PrimPol in TFV toxicity, I biochemically assessed a PrimPol active site mutant in an HIV+ patient taking TFV. The mutant appears to have drastically reduced polymerase activity and complete loss of priming activity, which may predispose this patient to TFV toxicity. With NNRTIs, there are continuous development efforts to improve pharmacokinetic properties and combat drug resistance. To this end, a series of 2-naphthyl phenyl ether compounds were developed to target the Y181C mutation of RT. Interestingly, early structures of RT with these class of compounds showed two different binding modes that affected potency against the mutant. By solving structures of 2-naphthyl phenyl ether derivatives with WT and Y181C RT, we determined that the compounds that interact with W229 retain potency against the mutant. These studies will be important to consider in the development process of next generation NNRTIs. Polα, in complex with Primase, is similar to PrimPol by possessing the ability to carry out de novo synthesis of nucleic acid primers. The primary role of the Polα-Pri complex in the primosome is to produce Okazaki fragments during DNA replication in a coordinated manner. Where primase initiates the primer with ribonucleotides, Polα continues the initial primer with deoxyribonucleotides. Interestingly, recent evidence shows that after replication mutations are left over from Polα, which is low-fidelity and lacks a proofreading mechanism. To gain insight on Polα’s activity during replication, we solved the structure of Polα with two replication-like substrates (Polα:dNTP:RNA/DNA or DNA/DNA) and kinetically characterized its activity with these substrates. We observed that with the RNA/DNA structure, a kink in n-4 sugar on the RNA primer correlated to a decrease in activity of the enzyme. Our kinetic characterization also revealed that with the DNA/DNA strand, Polα had increased incorporation efficiency but lower processivity. Our studies provide evidence of how different nucleotide substrates may regulate polymerase activity during replication. Taken together, the studies of three different polymerases presented here provide a mechanistic and functional understanding of these polymerases in diseases and potential treatments. Ultimately, these findings will contribute to the development of therapies in diseases where polymerases play a vital role.
Duong, Vincent Nghi, "Mechanistic insights into the roles and activities of polymerases in host and viral replication" (2021). Yale Graduate School of Arts and Sciences Dissertations. 41.