Structural and functional studies of antiviral drug toxicity
Antiviral drugs based on nucleoside analogs are effective inhibitors for viral reverse transcriptase and RNA polymerase, thus have been successfully used in treating HIV and HCV infections. With prolonged patients life span, the success of the drugs now has to be balanced with their drug toxicity. One of the major target of nucleoside analogs is human mitochondrial DNA polymerase, Pol Y. Because drug efficacy is not completely correlated with drug toxicity, we believe there is exploitable difference in designing potent, low toxic antiviral reagents. To reveal the structural differences between viral target protein and human adverse reaction target, we embarked on structural and functional studies of replicating human mitochondrial DNA polymerase or stalled by antiviral drugs. My laboratory determined the first crystal structures of human Pol Y holoenzyme. Recently, we determined structures of ternary complex of Pol Y-DNA with a substrate or an antiHIV reagent, zalcitabine, lamivudine or emtricitabine. These structures provided unprecedented insight in Pol Y mediated antiviral drug toxicity. As Pol Y mutations are associated with multisystem disorders, the structures have been widely used by basic scientists as well as clinicians to understand the detrimental effects of the mutations. I directed all of these studies.
Mitochondrial DNA repair
Mitochondria contain high concentrations of reactive oxygen species (ROS) due to intrinsic radicals generated through metabolic reactions and extrinsic factors such as anticancer radiation therapy. Consequently, mitochondrial DNA suffers higher likelihood for oxidative damages than chromosomal DNA. While the overall scheme follows that of nuclear BER, mitochondrial BER has distinct differences. Pol Y is responsible for DNA synthesis during replication and repair. I lead investigation of Pol Y activity in BER specific gap-filling DNA synthesis. Our findings indicate Pol Y is very inefficient on 1-nt gapped DNA and no strand displacement synthesis activity, suggesting that the polymerase alone is inefficient to carry out mitochondrial BER function, supporting the importance of repair complex. We studied Pol Y replication on damaged DNA. We recent started structural and functional studies of components of mitochondrial DNA repair complex with a long-term goal of structural determination of the entire mitochondrial DNA repairsome. I am the PI of these studies.
- Lee, YS., Kennedy, PW., and Yin, YW., “Structural insights into human mitochondrial DNA replication and disease-related polymerase mutations”, 2009, Cell, 139(2):312-324
- Lee, YS., Molineux, IJ., Johnson, KA., Yin, YW., “A single mutation in human mitochondrial DNA polymerase Pol gamma affects both polymerization and proofreading activities”, 2010, J. Biol Chem, 285(36):28105-28116.
- Lee, YS., Lee, S., Delemer B., Molineux, IJ., Johnson, KA., Yin, YW., “Each monomer of the dimeric accessory protein for human mitochondrial DNA polymerase has a distinct role in conferring processivity”, 2010, J Biol Chem, 85(2):1490-14999
- Sohl, CS., Szymanski, MR., Mislak, AC., Shumate, CK., Amiralaei, S., Schinazi, RF., Anderson, KS., Yin, YW., “Probing the Structural and Molecular Basis of Nucleotide Selectivity by Human Mitochondrial DNA Polymerase γ, 2015, PNAS, 112(28):8596-601.
- Szymanski, MR., Kuznestov, VB., Shumate, CK., Meng, Q., Lee, YS., Patel, G., Patel, S., Yin, YW., “Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase”, 2015, EMBO J, 34(14):1959-70.