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Author

Kelly Crowley

Date of Award

Spring 2011

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. Michelle L. Hamm

Abstract

In normal basepairing of DNA, the 2’-deoxyguanosine (dG) nucleotide will only pair with 2’-deoxycytidine (dC). Reactive oxygen species produced constantly by radiation, chemical carcinogens and as byproducts of respiration can oxidize the C8 position of dG, forming 8-oxo-2’-deoxyguanosine (OdG). OdG can then form stable base pairs with both dC and 2’-deoxyadenosine (dA). When a dA opposite OdG is replicated, it will be paired to dT rather than dG, causing a dG→dT mutation in the DNA code. These mutations have been linked to diseases such as arthritis, lupus and cancer. We sought to gain insight into OdG replication with different polymerase types. Klenow Fragment-exo (KF-exo) from E. coli, a type A polymerase, and Archaebacteria DNA polymerase IV (Dpo4), a type Y polymerase, were tested for their ability to incorporate dCTP or dATP opposite dG, OdG and various analogues. The analogues for dG and OdG differed in the imidazole ring and included CldG and BrdG, which mimic dG at N7, contain increasing steric bulk at the C8 position similar to OdG, and have diminished hydrogen bonding abilities at C8, as well as SdG and CdG, which similarly contain atoms of various size at C8 and diminished hydrogen bonding ability at C8, but mimic OdG at N7. Each analogue was tested with KF-exo and Dpo4 in order to better understand the exact steric and electronic properties that dictate the incorporation of dCTP and dATP opposite OdG with these different polymerase types.

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