The genetic material of all organisms is made up of a macromolecule called DNA. DNA is essential for cell survival as it encodes the genetic information for cells to properly function. However, DNA is exposed to both endogenous and exogenous agents, which can cause damage to the DNA leading to alterations within the genetic code. The alterations are known as mutations, which are responsible for an array of diseases that include cancer. Sources of DNA damage inducing agents include environmental pollutants, and free oxygen radicals produced during cellular metabolism. Specifically, acetylaminofluorene (AAF) and Benzo[a]pyrene (BaP), which are present within the environment, and are relatively inert. However, once metabolized within our cells the compounds are then transformed into their reactive forms N-Acetoxy-2-acetylaminofluorene (AAAF) and Benzo[a]pyrene, 7-8, dihydrodiol, 9-10, epoxide (BPDE). These activated products modify DNA by covalently bonding to guanine predominately at the N2 position. The presence of these adducts interfere with enzymes that are involved in DNA replication ultimately leading to cell death. In order to prevent cell death our cells contain specialized enzymes known as Y-family polymerases, which are able to bypass sites within DNA, where adducts are present in an effort to continue the process of DNA replication. Human Y-family polymerase eta is speculated to bypass DNA damage caused by AAAF and BPDE. N2-dG AAAF adducted DNA was purified using Polyacrylamide gel electrophoresis. Reverse phase chromatography was used to purify N2-dG BPDE from undamaged DNA yielding four enantiomers. Data from the RP-HPLC and PAGE provide evidence that supports the purification of N2-dG AAAF and N2-dG BPDE adducts on a synthesized DNA template, once confirmed they will be used as DNA templates to study translesion synthesis (TLS) by polymerase eta in vitro.

Synthesis of DNA containing a major lung carcinogen, benzo[a]pyrene and a model liver carcinogen, N-acetyl-2-aminofluorene

247th National Meeting (2014)

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