DNA repair of a single UV photoproduct in a designed nucleosome

Eukaryotic DNA repair enzymes must interact with the architectural hierarch y of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chrom atin structures involved in regulating other DNA processing events. The het erogeneity of lesions induced by DNA-damaging agents has led us to design h omogeneously damaged substrates to directly compare repair of naked DNA wit h that of nucleosomes. Here we report that nucleotide excision repair in Xe nopus nuclear extracts can effectively repair a single UV radiation photopr oduct located 5 bases from the dyad center of a positioned nucleosome, alth ough the nucleosome is repaired at about half the rate at which the naked D NA fragment is. Extract repair within the nucleosome is > 50-fold more rapi d than either enzymatic photoreversal or endonuclease cleavage of the lesio n in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extr acts, even in the presence of a large excess of undamaged DNA. This is an e xample of selective nucleosome assembly by Xenopus nuclear extracts on a sh ort linear DNA fragment containing a DNA lesion.