We have investigated the effects of DNA damage by (؎)-anti-benzo[a]pyrene diol epoxide (BPDE) and UV light on the formation of a positioned nucleosome in the Xenopus borealis 5S rRNA gene. Gel-shift analysis of the reconstituted products indicates that BPDE damage facilitates the formation of a nucleosome onto this sequence. Competitive reconstitution experiments show that average levels of 0.5, 0.9, and 2.1 BPDE adducts͞146 bp of 5S DNA (i.e., the size of DNA associated with a nucleosome core particle) yield changes of ؊220, ؊290, and ؊540 cal͞mol, respectively, in the free energy (⌬G) of nucleosome formation. These values yield increases of core histone binding to 5S DNA (K a ) of 1.4-, 1.6-, and 2.5-fold, compared with undamaged DNA. Conversely, irradiation with UV light decreases nucleosome formation. Irradiation at either 500 or 2500 J͞m 2 of UV light [0.6 and 0.8 cyclobutane pyrimidine dimer͞146 bp (on average), respectively] results in respective changes of ؉130 and ؉250 cal͞mol. This translates to decreases in core histone binding to irradiated 5S DNA (K a ) of 1.2-and 1.5-fold compared with undamaged DNA. These results indicate that nucleosome stability can be markedly affected by the formation of certain DNA lesions. Such changes could have major effects on the kinetics of DNA processing events.

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.