Strong evidence exists that exocyclic DNA adducts accumulate as end products of a complex cascade of events originating with diverse factors such as genetic diseases, diet, infections, and environmental toxicants. These exocyclic DNA adducts arise from exposure to bifunctional electrophiles and include single base adducts, intra and interstrand crosslinks, as well as DNA-protein crosslink. To develop a comprehensive understanding of the biological consequences associated with the formation of these lesions, it is necessary to characterize cellular responses, including replication bypass, repair and mutagenesis. It is hypothesized that a subset of exocyclic adducts, formed from reaction of hydroxyalkano species with guanine, adenine and cytosine, will be capable of undergoing transient ring-opening leading to formation of these complex lesions. To address these issues, a series of interrelated aims are proposed.
Specific aim 1 addresses the biological consequences of replicating DNAs containing exocyclic adducts, in which eukaryotic and prokaryotic shuttle vectors containing site and stereospecific exocyclic adducts will be assayed for their mutagenic potential. Since it is hypothesized that the proximal, but not the distal hydroxyalkano adducts will form various crosslinked species in a highly sequence context-dependent manner, a strategy has been developed in specific aim 2 to detect secondary spontaneous chemical reactions of 2-and 3-carbon heterocyclic DNA adducts that could produce intra and interstrand DNA crosslinks.
Specific aim 3 expands the type of DNA lesions being investigated to include hydroxyalkano DNA-protein crosslinks. Strategies have been developed that allow the creation of DNAs containing site-specific DNA-protein adducts, in which the linkage is either through secondary reactions with the ring-opened forms of the exocyclic DNA adducts or through the Cl' of the deoxyribose sugar and a primary amine on a DNA glycosylase/AP lyase. By achieving these aims, it will be possible to develop a comprehensive understanding of the biological implications of the reaction of DNAs with bifunctional electrophiles.
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