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Date of Award

Spring 2011

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science



First Advisor

Dr. Michelle L. Hamm


8-oxo-2’-deoxyguanosine (OdG), a mutated form of the DNA nucleotide 2’ deoxyguanosine (dG), is formed when dG is oxidized at the C8 position by reactive oxygen species. dG base pairs with 2’-deoxycytosine (dC), promoting correct DNA replication, but OdG can base pair with both dC and 2’-deoxyadenosine (dA). If OdG is paired with dA, the subsequent round of replication results in a point mutation, specifically a dG to dT transversion. The DNA polymerase selectivity controls whether dC or dA incorporation is preferred opposite OdG, and the frequency of incorporating dA determines mutation frequency. This study focuses on the large fragment of DNA polymerase I from Bacilius stearothermophilus (BF) and its dCTP:dATP incorporation ratio of 1:9. It has been predicted that this ratio is seen due to both steric hindrance caused by the C8 oxygen of OdG when bound to dC in the anti conformation, and a stabilizing hydrogen-bond between the sidechain of Q797 on the enzyme and the C8- oxygen of OdG when bound to dA in the syn conformation. To further study the observed incorporation ratio with BF, analogues of OdG were synthesized, incorporated into DNA, and used in experiments along with dG and OdG. This work indicates that the low efficiency of dCTP incorporation opposite OdG is not simply due to sterics; CldG and BrdG, which contain larger atoms off C8 than OdG, directed more efficient incorporation of dCTP than OdG. Also, the hydrogen bond between the enzyme and nucleotide may not be absolutely required for dATP incorporation, since that opposite CdG (which cannot form such a hydrogen bond) was similar to that opposite OdG. Previous studies have also focused on the incorporation of OdGTP opposite dC or dA, and have shown that this efficiency also depends on the enzyme. To study this further, the triphosphates of the analogues were synthesized (the synthesis of SdGTP was developed in this study) and some preliminary studies of incorporation opposite dC or dA were performed with different polymerases.

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