DYNAMICS OF DNA DAMAGE RECOGNITION BY REPAIR ENZYMES

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DESCRIPTION: The long term goal of this proposal is to develop a molecular understanding, derived from a combined theoretical-experimental collaborative approach, of the principles of specificity of the recognition of damaged DNA by repair enzymes and the stability of the enzyme-DNA complex. Recent x-ray determination of the structure of several repair enzymes presents an opportunity to investigate in molecular details the mechanisms of selective recognition of DNA damage. The researchers will investigate the recognition of DNA with a thymine dimer lesion that is recognized by the enzyme T4 endonuclease V. The availability of the x-ray structure of the enzyme and the complex with damaged DNA is the impetus for the investigation of the molecular principles of the specificity and stability of the complex. The working hypothesis is that four factors contribute to selective recognition of damaged DNA. These are encoded in 1) changes in the structure of DNA around the damaged area, 2) the ability of DNA to undergo a spontaneous or protein-induced flipping of a base near the damaged site into an extrahelical position, 3) asymmetric distribution of condensed counterions around damaged DNA that produces an electrostatic orientational field, and 4) changes in DNA hydration around the point of damage that influences the thermodynamics of the interaction between the repair enzyme and the damaged DNA. These principles appear to be generally applicable to many repair enzymes recognizing damaged DNA. The aims of this proposal to investigate in detail these factors, cannot be fully accomplished by either an exclusively theoretical or a purely experimental approach and they, therefore, propose a complementary collaborative approach in which they combine computational simulations with time-dependent fluorescence measurements to investigate the factors that determine the specificity of damaged DNA recognition by repair enzymes. Interpretation of experimental results will be supported by the realistic representation of molecular models, their vibrational properties and the aqueous ionic environment derived from molecular simulations. In a complementary way, mechanistic interpretations and predictions from molecular simulations will be tested rigorously by experimental measurements. Such complementary research will lead to a better understanding of the factors that contribute to specificity of repair enzymes and play an important role in specific protein-DNA interactions in general.