Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome

A-175-base pair fragment containing the Xenopus borealis somatic 5 S riboso mal RNA gene was used as a model system to determine the effect of nucleoso me assembly on nucleotide excision repair (NER) of the major UV photoproduc t (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extra cts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD s ites in the 5 S rDNA fragment while having little effect at a few sites. Th e time course of CPD removal at 35 different sites indicates that >85% of t he CPDs in the naked DNA fragment have t1/2 values <2 h, whereas <26% of th e t1/2 values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyed. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition.