||Oxidized guanine, a type of DNA damage, pairs with adenine or cytosine, creating the potential for mutations. The DNA repair enzyme MutY is able to identify this mispairing of the oxidized guanine and adenine and subsequently remove the adenine. MutY contributes to the overall program of Base-Excision Repair (BER), which is important in many human genetic diseases. We believe that we have found genes corresponding to MutY in microbes living in the Lost City, a thermal field in the mid-Atlantic with extreme conditions that are thought to resemble conditions at the beginning of life. However, these Lost City MutY genes may be incomplete, missing the portion which encodes the C-terminal, OG-recognition domain. ; In order to test the activity of the Lost City MutY samples, I created chimeras for a DNA repair enzyme with the N-terminal domain derived from microbes in the mid-atlantic and the C-terminal domain from a thermophilic bacterium, Geobacillus stearothermophilus. By adding a known and complete C-terminal, OG-recognition domain, these Lost City MutY chimeras can be tested to see if they still retain their function of repairing DNA. The DNAs encoding N-terminal portion of MutY from the Lost City candidates, the C-terminal domain from Geobacillus stearothermuphilus, and the pkk223 vector DNA were assembled through ligation-independent cloning (LIC). LIC involves amplification through PCR, purification through agarose-gel electrophoresis, digestion by DpnI, which destroys the PCR templates, and transformation to generate bacteria harboring the assembled MutY chimera-encoding plasmid DNA. Purified DNA plasmids were sent for sequencing to check the accuracy of the MutY chimera clones. With this, the chimeras can now be tested using a Mutation Rate Assay which will determine the DNA repair function of each Lost City MutY. Presented at ACCESS Symposium at the University of Utah.