) During the current funding period, we have applied spectroscopic and calorimetric techniques to characterize the thermal (delta Go, delta Ho, delta So, delta Cp) and extra-thermodynamic impacts of mutagenic lesions on the stabilities, conformational preferences, temperature-dependent transitions, and melting cooperativities of DNA duplexes. Our program for the requested funding period is divided into two parts, to be pursued in parallel. In Part I, we will build on our embryonic database for lesion-containing DNA duplexes by evaluating: (i) the communication of lesion effects through the DNA backbone and/or stacked bases; (ii) the impact of lesions on the length and flexibility of DNA; (iii) a new fluorescence-based methodology for rapid and reliable determination of DNA duplex stabilities; and (iv) the impact of additional lesions on the physicochemical properties of DNA duplexes, particularly interstrand crosslinks, FapydG, the major acrolein adduct of guanine, and the carbocyclic analogs of dA, 8-oxodG and the abasic site. The effects of sequence context (cross-strand partner bases and flanking bases) on the thermodynamic and extra-thermodynamic impacts of these lesions will be assessed with an eye towards further evaluating the role of """"""""energetic discrimination/recognition"""""""" of structurally similar DNA domains. In Part II of our program, we will use calorimetric methods (i) to define the energetic landscape of template-directed DNA synthesis by an exonuclease deficient Klenow fragment of E. coli polymerase I and (ii) to characterize the thermodynamics of repair protein recognition of damage in DNA. These efforts are designed to define lesion-induced physicochemical differences that may provide the basis for selective recognition of damaged DNA sites by the machinery of repair. Our ultimate goal is to use our spectroscopic and calorimetric results, together with NMR studies, to define relationships between structure, energetics, and biological activity. Such correlations should help us elucidate mechanisms of chemical and radiation induced mutagenesis, lesion recognition, and repair.
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