Pelloux Julie, Tremblay Maxime, Wellinger Raymund J, Conconi Antonio
Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC, Canada.
Methods Mol Biol. 2012;809:303-20. doi: 10.1007/978-1-61779-376-9_21.
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.
环丁烷嘧啶二聚体（CPDs）和（6,4）嘧啶 - 嘧啶酮二聚体是紫外线诱导产生的主要DNA损伤（或光产物），并通过核苷酸切除修复（NER）途径被清除。如果不进行修复，DNA损伤会导致基因组不稳定。基因组被组织成具有不同功能和染色质结构的核区域。尽管对所有染色体环境中的NER进行研究对于理解基因组维持机制很重要，但我们专注于核仁中的NER。核糖体DNA（rDNA）位点的吸引人之处在于其染色质结构；并非所有rRNA基因都被转录，活跃（无核小体）和不活跃（有核小体）的rRNA基因在核仁中共存。这些特征使得能够直接比较两种非常不同的染色质结构中的NER。酵母被用作模型系统，采用的方法如下：细胞核分离、用限制酶消化染色质以释放活跃的rRNA基因、补骨脂素交联、用T4内切核酸酶V检测CPDs以及在相对大片段DNA上检测CPDs修复，以及用引物延伸在核苷酸水平追踪DNA损伤和修复。使用这种方法，我们已经表明，与核小体rDNA相比，非核小体中的NER更快，CPDs的形成促进了活跃rDNA染色质的变化，并且NER与rRNA基因转录相关联。
