Session: Current Topics in Biological Chemistry:

Oxygen, required by aerobic organisms to sustain life, also yields reactive oxygen species (ROS) which cause oxidative damage to nucleobases in DNA. This oxidative damage results in 8-Oxoguanine (8-OG), a common oxidative product which mispairs with adenine due to its thymine-resembling Hoogsteen base pair face. Such 8-OG:A mispairs cause G:C→T:A transversions upon DNA replication, a build-up of which is destructive to the cell. MutY, a base excision repair (BER) enzyme, combats oxidative damage to guanine nucleobases in DNA by cleaving the mispaired adenine nucleobase. This cleavage results in an abasic site across from 8-OG, which is later repaired by downstream BER enzymes. Inherited variants of MUTYH, the human homolog, have been linked to the initiation and development of carcinogenesis. How MutY is able to differentiate the mispaired adenine from correct T:A pairs is the focus of my research in the David Lab. Discovering critical structural features, like which amino acids are primarily responsible for MutY recognition, will aid in the development of inhibitor or activator molecules with therapeutic applications. Prior work by the David Lab showed the N2-exocyclic amino of 8-OG in DNA is critical for repair by MutY. This work will determine the effect of steric bulk and hydrogen bonding imparted by the exocyclic amino of 8-OG on the target recognition mechanism of MutY via 8-Oxo-2’-dG analogs modified at the 2-position, and will analyze the duplex stability of produced oligonucleotides. The use of our synthetic analogs as substrates of MutY will reveal the structural features dominant in MutY’s target recognition of the 8-OG:A mispair.

Previously unreported N2-Ethyl-, N2,N2-Diethyl-, and N2,N2-Dimethyl-8-Oxo-2’-dG phosphoramidite analogs have been synthesized and confirmed. Numerous DNA sequences of the N2-Ethyl-, N2,N2-Dimethyl-, and N2,N2-Diethyl-8-Oxo-2’-dG oligonucleotides have been purified and confirmed. Furthermore, the results of the herein reported duplex stability studies support the viability of produced oligonucleotides for use as substrates in enzymatic assays; future work will involve EMSA-based and cellular-based assays using MutY and reported oligonucleotides, and further examination of duplex stability will be conducted.

Sheila David

Univ of California

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