Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site

A strategy was developed to assemble nucleosomes specifically damaged at on ly one site and one structural orientation. The most prevalent UV photoprod uct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesiz ed and incorporated into a 30 base oligonucleotide harboring the glucocorti coid hormone response element. This oligonucleotide was assembled into a 16 5 base pair double stranded DNA molecule with nucleosome positioning elemen ts on each side of the cs CTD-containing insert. Proton NMR verified that t he synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut similar to 90% o f the dsDNA molecules. This cleavage is completely reversed by photoreactiv ation with E. coli UV photolyase, further demonstrating the correct stereoc hemistry of the photoproduct. Nucleosomes were reconstituted by histone oct amer exchange from chicken erythocyte core particles, and contained a uniqu e translational and rotational setting of the insert on the histone surface . Hydroxyl radical footprinting demonstrates that the minor groove at the c s CTD is positioned away from the histone surface about 5 bases from the nu cleosome dyad. Competitive gel-shift analysis indicates there is a small in crease in histone binding energy required for the damaged fragment (Delta D elta G similar to 0.15 kcal/mol), which does not prevent complete nucleosom e loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosom es can be experimentally designed for in vitro DNA repair studies.