The broad objective of this proposal is to understand the molecular details of an important defense mechanism (DNA) repair) against phenotypic changes and mutations induced by insults to DNA in human cells. Such changes are an important etiological factor in cell survival and cancer. These insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of the majority of these insults occurs via the """"""""long path"""""""" excision repair mechanism, UV radiation will be used as the prototype environmental agent for these studies. In addition, repair of bleomycin-induced DNA damage, which mimics damage by ionizing radiation, will be studied to elicit an important alternative (short patch) excision repair mode which requires less processing at the chromatin level. Yeast minichromosomes will be used as a model chromatin substrates to study efficiency and completion of repair of UV photoproducts in well-defined structural domains in both repair deficient yeast cells and human cell extracts. These studies will employ a novel Southern blot analysis to follow repair at specific sites in different chromatin structures, and dThd uptake mutants to follow completion of repair in intact yeast cells. Secondly, the composition of nascent and mature repair sites in human chromatin will be analyzed using specific """"""""tagging"""""""" of repair sites with nucleotide analogues and specific antibodies to modified histones; while the structure of these sites will be analyzed using Hg-affinity chromatography. Thirdly, high resolution mapping of UV photoproduct formation and RNA polymerase blockage will be examined in a positioned nucleosome in vitro. Finally, repair of bleomycin-induced DNA damage will be assessed in specific genes in human chromatin using a modification of the standard method for measuring UV photoproduct repair. Thus, we will use a """"""""multifaceted"""""""" approach to examine the role of chromatin structure in DNA damage and its repair with the ultimate goal of understanding this complex process in human cells. Since DNA lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.
Showing the most recent 10 out of 34 publications
