CARCINOGENS AND CHROMATIN STRUCTURE AND FUNCTION

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The poly ADP-ribosylation modification of nuclear proteins is the initial cellular response to DNA damage caused by chemicals. Poly(ADP-ribose) polymerase (PADPRP) catalytic activity is absolutely dependent upon DNA and its activity is directly correlated with the number of strand breaks in DNA. Thus, when cells, are exposed to chemical carcinogens, there is an immediate drop in cellular NAD and concomitant rapid and transient increase in poly ADP-ribosylated nuclear proteins. Recent progress on this project involved the cloning, sequencing, and hyperexpression of transfected genes for this enzyme in both human and murine systems. Preliminary data using transient transfection indicated that cells hyperexpressing PADPRP have increased rates of DNA repair. We also were able to map the precise chromosomal loci for this gene in both human and mouse. During the renewal period, we intent to utilize the molecular and biochemical data obtained to better clarify and characterize the role of ADP-ribosylation in DNA repair mechanisms. Methods in Aim I utilize a variety of molecular approaches including antisense and ribozyme expression, to experimentally modulate ADP-ribosylation potential in cells subjected to DNA strand breaks and assess the effects of this by utilizing new methods for precisely measuring preferential gene repair and recombinase activity using novel plasmid assays. Nuclear targets for ADP ribosylation are the focus of Aim II. The new molecular information will be used here to relate structure of PADPRP (and its nuclear protein acceptors) to function. For example, c-fos is a nuclear acceptor for PADPRP and also transcriptionally activated by strand- breaking chemicals. PADPRP activity modulated by antisense will be used to address the biological function of the PADPRP of fos, and as an example of approaches to be used for other nuclear protein acceptors during the renewal period.