Energetics represents the missing link between structure and function. As such, characterizing the energetic impacts of lesions on duplex properties and on protein recognition/binding events is crucial for understanding the influences of lesions on DNA replication, mutagenesis, and repair pathways. These characterizations are particularly important since we have demonstrated that profound lesion-induced alterations in duplex energetics can occur even in the absence of significant structural changes, raising the intriguing possibility of "energetic recognition". During the requested funding period, we will use a combination of spectroscopic and calorimetric techniques to characterize the impacts on duplex properties of 8-oxo-dG; abasic sites; bistrand abasic lesions; Fapy-dG and thymine glycol sites, including their carbocyclic derivatives; template misalignment defects (e.g. bulged structures); and defects originating from replication errors (e.g., bulges, abasic bulges, single base substitutions), which have been associated with cancer and other diseases. We also will map the energetic landscape of the base excision repair pathway by characterizing the energetics of repair enzyme binding to their potential substrate, transition state, intermediate state, and product analogs. Specific repair enzymes targeted for study include two purine glycosylases, Fpg and Ogg1 (a mammalian functional homolog of Fpg), as well as two pyrimidine glycosylases, E. coli endo VIII (a structural homolog of Fpg called Nei) and E. coli Endo III. Many of these studies will be conducted using nonhydrolyzable carba analogs to evaluate binding in the absence of turnover. We will use isothermal titration and stopped-flow mixing calorimetry to characterize the impact of lesions on polymerase binding and template-directed DNA synthesis, thereby elucidating the energetic origins of DNA polymerase fidelity, including misincorporation of bases that occurs during translesion synthesis. In conjunction with the parallel structural (Project 2) and biological studies (Project 1) being pursued as part of this program project, our proposed energetic characterizations of lesions on duplex properties, repair enzyme recognition, and template-directed DNA synthesis will allow us to define microscopic/macroscopic/functional correlations that no single approach alone could yield. This integration of insights derived from multiple experimental platforms will enable us to better understand mechanisms underlying fundamental processes involved in oxidative DNA damage, including lesion formation, DNA repair, and mutagenesis.