STRUCTURE OF DNA LESIONS IN SUPERHELICAL DOMAINS

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DNA repair systems recognize a multitude of lesions in DNA, including bulge defects, non-homologous regions, cyclobutane dimers, and the presence of DNA-damaging adducts. Efficient recognition and repair of such lesions occurs despite the presence in the cell of a wide range of normal heterogeneities in DNA structure. It has been proposed that these lesions cause significant alterations of DNA structure and dynamics; however, nearly all of what is known about these defects in DNA structure is based on studies that have been carried out on linear DNA molecules in the absence of superhelical stress. Recent work from this laboratory on the structure of intrinsically bent and unbent A-tract containing DNA sequences indicates that the sequence-dependent structure of these tracts in linear DNA fragments cannot be extrapolated to super coiled domains. Dramatic changes in DNA super coiling that occur during transcription raise important questions about the effect of super coiling on defect structure and its role in transcription-couple DNA repair. The objective of this work is to investigate the conformation and dynamics of several DNA-structure defects in superhelical domains as models of DNA lesions recognized by DNA repair systems. The tertiary structure of super coiled molecules containing bulges (insertion/deletion modification), mismatches, and covalent adducts of psoralen and the antineoplastic drug cisplatin will be examined by site-specific recombination and conventional and cryoelectron microscopy in conjunction with computer simulation. The distribution of knots and catenanes generated by bacteriophage lambda integrative (Int) site-specific recombination is a reporter of the tertiary structure of the DNA substrate. Observed distributions of Int- recombination products will be analyzed using Monte Carlo computer simulations of intramolecular recombination in order to quantitate changes in DNA bending distortion and/or bending, flexibility. The conformation of super coiled, lesion-containing DNA molecules deduced from these solution studies will be compared with that observed by independent conventional and cryoelectron microscopy experiments.