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Author

Nicole Byram

Date of Award

2017

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. Michelle Hamm

Abstract

8-oxo-2'-deoxyguanosine (OdG) , a promutagenic lesion, arises from a reaction between 2'-deoxyguanosine (dG) and reactive oxygen species (ROS) such as peroxides, hydroxy radicals, and superoxides. ROS are produced from environmental carcinogens, radiation exposure, and metabolic respiration. OdG can base pair with both 2'-deoxycytidine (dC) and and 2'­ deoxyadenosine (dA), the latter of which causes dG to thymidine (dT) transversions. These mutations may be responsible for the link between OdG and aging, cancer, lupus, and

rheumatoid arthritis. To examine the preference for dCTP and dATP incorporation opposite OdG, the A-family polymerase Klen-Taq-1 (KT) was studied. KT is a Kienow-Fragment (KF) analog of Taq DNA polymerase from Thermophilus aquaticus and is more stable than KF while also lacking a 3'- exonuclease site. Previous studies have determined that potential steric clashing between a major groove amino acid and the C2- and C8- positions of OdG is critical in determining the efficiency of dCTP or dATP incorporation. KT (like KF) possesses a methionine in the major groove (Met672) rather than the isoleucine that is found in A-family polymerase I from the bacterium Bacillus Stearothermophilus (BF). The activity of dCTP and dATP incorporation opposite OdG and seven of its analogues was examined with wild-type KT and compared with wild-type BF to study major groove steric clashing with the C2- and C8- positions and their importance to OdG mutagenicity.

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