UV-induced DNA damage and DNA repair in ribosomal genes chromatin

Methods Mol Biol. 2012;809:303-20. doi: 10.1007/978-1-61779-376-9_21.

Abstract

Cyclobutane pyrimidine dimers (CPDs) and (6,4) pyrimidine-pyrimidone dimers are the major DNA lesions (or photoproducts) induced by ultraviolet light and are removed by the nucleotide excision repair (NER) pathway. If not repaired, DNA damage can lead to genome instability. The genome is organized into nuclear domains with distinct functions and chromatin structures. Although studies on NER in all chromosomal contexts are important to understand the mechanisms of genome maintenance, we focused on NER in the nucleolus. The attractive feature of the rDNA locus is its chromatin structure; not all rRNA genes are transcribed and both active (no nucleosomes) and inactive (nucleosomes) rRNA genes coexist in the nucleolus. These characteristics allow for direct comparison of NER in two very different chromatin structures. Yeast is used as a model system and the methods employed are as follows: nuclei isolation, restriction enzyme digestion of chromatin to release active rRNA genes, psoralen cross-linking, T4-endonuclease-V enzyme to detect CPDs and CPDs repair over relatively large stretches of DNA, and primer extension to follow DNA damage and repair at nucleotide level. Using this approach, we have shown that NER is faster in nonnucleosomes vs. nucleosomes rDNA, that the formation of CPDs promotes changes in the active rDNA chromatin, and that NER is coupled to rRNA genes transcription.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Southern
  • Chromatin / metabolism
  • DNA Damage / genetics
  • DNA Damage / radiation effects*
  • DNA Repair / genetics
  • DNA Repair / physiology*
  • DNA, Ribosomal / genetics*
  • Electrophoresis, Agar Gel
  • Ficusin / chemistry
  • RNA Polymerase I / metabolism
  • Ultraviolet Rays*

Substances

  • Chromatin
  • DNA, Ribosomal
  • RNA Polymerase I
  • Ficusin