The repair of DNA damage is critical is critical for genome stability in any organism. In all eukaryotic cells, DNA excision repair must overcome the obstacle of nucleosomes that are complexed with the DNA to form chromatin. These nucleosomes must somehow rearrange in order to allow passage of the repair proteins. The proposed research project would investigate the nucleosome """"""""unfolding"""""""" model during excision repair of DNA in human fibroblasts. The first approach will directly examine the conformation of nucleosomes at nascent repair sites using organomercurial affinity chromatography, which separates """"""""open"""""""" nucleosomes from """"""""closed."""""""" The second approach involves analyzing sites excision repair for histone modifications, acetylation and poly-ADP ribosylation, which are both associated with open chromatin and appear to play a role in repair. Repaired chromatin will be isolated via a biotin tag on the nucleotides incorporated during the repair process by utilizing an avidin-cellulose column. The histones associated with this chromatin will be analyzed for modifications by electrophoresis. Immunoprecipitation and analyzing the amount of associated repair. The third approach implements an in vitro repair system to address rates of excision repair with acetylated or unfolded nucleosome substrates.
