Novel Base Excision Repair and Hypoxanthine in Live Human Cells
DeVito, Stephen Roy
Elucidating the mechanism of DNA damage and mutation is key to understating cancer genesis. In the first section of this thesis we examined base excision repair enzymes (BER) responsible for mitigating damage to DNA. BER DNA glycosylase activity was categorized both in silico, and in vitro. N-methylpurine DNA glycosylase (MPG) and 8-oxoguanine (OGG1) DNA glycosylases were shown to have novel substrate activity on 7,8-dihydro-8-oxoguanine (8-oxo-G) and hypoxanthine (Hx), respectively. We utilized surface plasmon resonance (SPR) and DNA glycosylase activity assays to characterize binding affinity and enzyme-substrate activity. We found the OGG1 bound to Hx with similar affinity as Hx’s canonical DNA repair enzyme MPG, yet produced little to no product. This led us to the hypothesis that Hx may have more than one DNA repair enzyme, and may also act as a potential replication blocker by binding and not releasing from DNA. To understand Hx’s effect on DNA repair we examined Hx repair in human colon cancer cell line HCT116 and normal human kidney fibroblast line HEK293. Here we found that Hx causes mutation in both leading and lagging strands of DNA, with an increase in mutation frequency found in the lagging strand. Further, Hx causes A-->G transition mutations, insertions, and deletions in both normal HEK293 and cancer HCT116 cells lines.
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