The long term goal of our research program is to elucidate the molecular mechanisms of DNA adduct-induced chemical carcinogenesis. Aromatic amines are well-known environmental human carcinogens. In particular, arylamine-DNA adduct formation has been confirmed in various human tissues and is believed to induce mutation. We have previously shown that arylamine adducts in DNA exist in three well-defined conformations: stacked (S), external B-type (B), and wedge (W). The conformation depends on the location of the carcinogen moiety in the DNA molecule, and the population ratios of the types under physiological conditions are sequence-dependent. In this application, we hypothesize that arylamine-induced repair and mutation is conformation-specific (S, B, W). We propose four major aims to help define adduct conformation and examine their specific effects on repair (initial damage recognition) and replication (replication fork heterogeneity). Specifically, these aims focus on: (1) conformation-specific repair in a human nucleotide excision repair (NER) system and long-range sequence effects, (2) damage recognition (protein-DNA interaction) (3) the thermodynamics of sequence-dependent slippage-induced frameshift mutagenesis, and (4) replication fork conformational heterogeneity and polymerase binding. We will employ not only existing dynamic 19F NMR/CD, EMSA and fluorescence spectroscopy, but also innovative chip-based surface plasma resonance (SPR) and differential scanning calorimetric (DSC) procedures, creating a powerful suite of biophysical methodologies. Successful completion of the proposed aims will help us gain a better grasp on the protein-DNA interactions involved in human NER and trans-lesion synthesis, which have important implications for resolving the molecular details of cancer etiology. Such knowledge will also be of help in the development of sensible prevention and risk assessment strategies.
The primary causes of sporadic human cancers are environmental. Aromatic amines are among the most notorious environmental chemicals that are implicated in the etiology of human cancers. Formation of arylamine-DNA adducts has been confirmed in various human tissues and is believed to induce chemical carcinogenesis. Hence it is imperative to elucidate how these lesions are repaired and replicated in vivo at the atomic and molecular levels. The key molecular players (adduct structures, polymerases, repair proteins) producing adverse outcomes must be identified, characterized, and understood in order to devise appropriate prevention and risk assessment strategies.
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