Date of Completion
Cyclopurines, DNA Damage, Translesion Synthesis, Enzyme Kinetics, Protein Purification, Dpo4, Polymerase IV
Dr. Ashis Basu
Dr. Steven Suib
Dr. Mark Peczuh
Field of Study
Doctor of Philosophy
Cyclopurines are tandem DNA lesions caused due to ionizing radiations. Tandem lesions can lead to genomic mutations if bypassed unfaithfully by replicative polymerases. Cyclopurine lesions notably, 8,5’-cyclo-2’-deoxyadenosine (cdA) and 8,5’-cyclo-2’-deoxyguanosine (cdG) are found to be genotoxic as well as mutagenic. In this study, to evaluate their mutagenic potential I have measured the rate of insertion of dNTP’s opposite and extension past cyclopurines by three different polymerases, E. coli DNA polymerase I (KF (exo-)), Sulfolobus solfataricus DNA polymerase IV (Dpo4) and E. coli DNA polymerase IV (pol IV). I have found that model KF (exo-) stalled at the adduct and was unable to form full-length extension products under running start conditions. Additionally the steady state kinetic parameters establish error free replication of both S-cdA and S-cdG by KF (exo-). I have further investigated the behavior of a Y-family polymerase in the bypass of these lesions. It was observed that S-cdA and S-cdG resulted in a major block of replication for DinB subfamily polymerase, Dpo4. However, they were able to extend a small fraction of the primer to a full-length product. Steady state kinetic parameters for Dpo4 suggest S-cdA replication to be error free while S-cdG is more prone to erroneous replication. For S-cdA dTMP was incorporated preferentially compared to the other nucleotides. On the contrary, for S-cdG the wrong nucleotide dTMP was preferred over the right nucleotide dCMP. This study reinstates the strong blocking nature of S-cdA and S-cdG.
As it was noted that a Y-family polymerase could bypass the lesion it was interesting to compare it with other Y-family polymerases. In my studies, I have used a Y-family polymerase from E. coli, Polymerase IV. Due to the lack of commercial availability of this polymerase, its gene was engineered in a bacterial expression vector and the enzyme was subsequently purified. On performing in vitro extension studies with this enzyme it was observed that S-cdA and S-cdG were a major block for replication for pol IV. Steady state kinetic parameters for pol IV suggest S-cdA and S-cdG replication to be error free. Although, compared to the control template the extension ability of the lesion containing templates was 1000 fold less efficient. The above in vitro studies confirms the blocking nature of 8,5’-cyclopurines-2’-deoxynucleosides.
Pednekar, Varsha, "In vitro Translesion Synthesis of 8,5’- Cyclopurine-2’-Deoxynucleosides" (2014). Doctoral Dissertations. 582.