Nucleotide Excision Repair of the 5 S Ribosomal RNA Gene Assembled into a Nucleosome

Xiaoqi Liu,Michael J Smerdon

doi:10.1074/jbc.m002206200

Abstract

A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) 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 dyad. 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.

Highlights

In eukaryotic cells DNA is associated with histone proteins in the structural hierarchy of chromatin, required to package the enormous length of DNA into the small volume of a nucleus
Nucleosomes were reconstituted onto 5 S rDNA fragments by histone octamer exchange from chicken erythrocyte (CE) core particles isolated from H1/H5-stripped chromatin [25]
The CE histone octamers were reconstituted onto 5 S rDNA by exchange in high salt followed by stepwise dialysis

Summary

Introduction

In eukaryotic cells DNA is associated with histone proteins in the structural hierarchy of chromatin, required to package the enormous length of DNA into the small volume of a nucleus. Exonuclease III digestions were carried out with 9 ␮l (ϳ6 ng of 5 S rDNA) of naked 5 S rDNA or reconstituted nucleosomes and incubated with 100 units of exonuclease III (Roche Molecular Biochemicals) at 37 °C for different times in a buffer containing 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 3 mM MgCl2, and 1 mM 2-mercaptoethanol.

Results

Conclusion