Structural elucidation of DNA adducts is crucial to understanding the initiation of carcinogenesis and ultimately in designing preventative strategies. Arylamine carcinogens are implicated in the etiology of various human cancers. We hypothesize that: (a) arylamine-adducts in DNA exist in two prototype conformations, a base-displaced """"""""stacked"""""""" (S) and an external binding normal """"""""B-type"""""""" 03); (b) a delicate S/B conformeric balance is modulated by both the base sequence surrounding the adduct site and the nature of specific interactions in the active sites of replication and repair enzymes; and (c) the resulting conformational heterogeneity plays a critical role in determining the mutagenic outcomes. We will use the well-documented aminofluorene (AF)-induced S/B heterogeneity to correlate their propensity to undergo repair and replication. This will be accomplished by the use of 19FNMR coupled with fluorine-labeled AF-modified DNA in various sequence contexts, with or without the presence of a polymerase.
Specific aims are to (1) measure the S/B ratios of double or single/double stranded duplexes in various sequence contexts; (2) to measure the S/B ratios of deletion duplexes, whose 3'-next nearest flanking sequence to the lesion is altered; (3) to measure the S/B ratios of 18/9-mer template-primers, whose 5'-flanking sequences are varied, in the presence of the polymerase Klenow Fragment (KFexo-); and (4) to conduct site-specific mutagenesis of the sequences used in Aims 1 and 3. Circular dichroism and melting experiments will also be conducted to define further the adduct structure. The polymerase experiments proposed in Aim 3, in particular, examine the adduct conformation at the replication fork within the active site of a polymerase and may provide,for the first time, a systematic basis for conducting conformation- and adduct-specific mutagenesis in a simulated biological environment. The proposed research will introduce 19F NMR as a powerful structural biology tool in investigating the mechanisms of arylamine mutagenesis.
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