CELULAR PROCESSING OF DAMAGED DNA--ROLE IN ONCOGENESIS

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Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Using relatively short, defined DNA sequences containing well characterized lesions, we have begun to analyze the intragenomic "fine structure" of DNA repair in cultured cells. Having discovered that certain regions of the nuclear genome repaired more efficiently than others, we hypothesize that the efficiency of repair of damage in mammalian chromatin depends upon the type of lesion, its location in the genome and the functional state of the DNA at the site of the lesion. Such specificity may account for some of the profound differences soon in the carcinogenic responses of different tissues and of the same tissue in different organisms. Having developed assays sensitive enough to detect repair of several different lesions, including pyrimidine dimers and interstrand cross-links, in restriction fragments from specific regions of the genome, we will compare the rate and extent of repair in genes that differ levels of expression, time of replication, genomic location and function. Examples include protooncogenes and other inducible or developmentally activated genes such as those for metallothioneins, alpha fetoprotein, fetal and adult beta-globin and myosin heavy chain in differentiating myoblasts. Chromatin conformation and methylation levels will be assessed as possible determinants of proficient repair. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair might account for mutagenic changes related to carcinogenesis. Replication of defined nucleotide sequences containing damage will be studied to determine whether differential levels of replication occur in particular genomic domains and whether daughter-strand discontinuities occur in those sequences. Defined chimeric plasmids containing lesions at unique sites will be used to introduce genes into different genomic domains and to probe the specific features of damage processing the increase the frequency of stable transformation of human cells. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage in biologic end points such as survival, mutagenesis, and carcinogenesis.