Penny J. Beuning
David E. (David Edward) Budil
Date of Award
Master of Science
Department or Academic Unit
College of Arts and Sciences. Department of Chemistry and Chemical Biology.
chemistry, biochemistry, DNA Damage, DNA replication, dnaE, Pol III, polymerase
DNA adducts, DNA replication, DNA polymerases, Escherichia coli
Biochemistry | Cell Biology
Accurate DNA replication is paramount for survival of all organisms. High-fidelity DNA polymerases ensure correct geometry of newly-forming base pairs by utilizing a tight fitting active site that allows only the correct incoming nucleotide to bind. This is reflected in the low error rate of high-fidelity polymerases; only 1 error in over 103 to 105 nucleotides incorporated. However this high geometric specificity can be disrupted. DNA is constantly under assault by mutagens, carcinogens, and reactive metabolic products. These agents can form adducts on DNA that disrupt high-fidelity DNA polymerases and stall the replication fork. The newly discovered Y family of DNA polymerases can bypass these lesions and allow the replication fork to continue. I am interested in the factors that make the major replicative DNA polymerase in E. coli, the DNA pol III alpha subunit, a high-fidelity polymerase. We have built a homology model of the enzyme in complex with DNA. We have identified potential residues important for fidelity. These residues were mutated and the polymerase variants were assayed by primer extension analysis on damaged and undamaged DNA. Engineering a low-fidelity polymerase from a high-fidelity polymerase allows us to identify the factors controlling polymerase specificity. Our computational models and kinetic data help to reveal the factors important in replication of damaged DNA.
Sharma, Rajal, "Engineering Escherichia coli DNA Polymerase III α for translesion synthesis" (2010). Chemistry Master's Theses. Paper 16. http://hdl.handle.net/2047/d20000331
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