The broad objective of this proposal is to understand, at the molecular level, an important defense mechanism against potentially lethal, mutagenic and/or carcinogenic insults to DNA in human cells. Such insults result from a wide variety of known environmental agents. Our approach has been to examine the role of chromatin structure in the process of recognition and removal of DNA lesions by repair enzymes in normal and (partially) repair deficient human cells. In the present application, I have proposed to examine the efficiency of DNA repair within different subdomains of nucleosomes in both human cells and a model yeast minichromosome. We will use novel exonuclease mapping techniques to determine the distribution of repair synthesis and DNA lesions following different extents of repair. These studies should indicate if certain regions of nucleosomes are refractory to efficient repair, and if repair deficient human and yeast cells lack factors required for efficient repair in the various nucleosome subdomains. We will also examine nucleosome rearrangements associated with repair of lesions induced by """"""""short patch"""""""" agents (e.g., ionizing radiation and bleomycin) to determine if the reduced DNA processing during these events requires different structural rearrangements than """"""""long patch"""""""" repair. Secondly, we will use a reversible, permeable cell system to """"""""tag"""""""" newly inserted repair patches in human cells with biotinylated nucleotides. These tags will allow us to isolate newly repaired regions of chromatin for studies on their protein composition and determine the distribution of repair sites in the human genome by electron microscopy. We hope to eventually sequence and identify proteins in human cells associated with different stages of DNA repair, and which may be altered in repair deficient human cells. Thirdly, we will explore the possibility that modification of nuclear proteins plays an active role in the DNA repair response. We plan to use the biotin-tag system to determine the level of acetylation and ADP-ribosylation of histones and nonhistone proteins in newly repaired regions. Furthermore, we will examine the efficiency of DNA repair within nucleosome subdomains of chromatin regions containing acetylated histones. Finally, we will determine if nucleosome migration is facilitated by this modification event. The results of these studies should provide a clearer understanding of the role of environment agents in such important cellular responses as survival, mutagenesis, carcinogenesis and aging.
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