From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260 and.
From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260 and; the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea.
J Biol Chem. 2013 Jul 19;288(29):20918-20926. doi: 10.1074/jbc.M113.482257. Epub 2013 Jun 8.
Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.
核苷酸切除修复是人类细胞中从基因组 DNA 中去除主要 UV 光产物的唯一机制。在体外用人细胞无细胞提取物或纯化的切除修复因子的情况下，通过双切口以 24-32 个核苷酸长的寡聚物（名义上为 30 -mer）的形式从裸露 DNA 或核小体中去除损伤。以前不知道 DNA 损伤是否以类似的方式从染色质中去除，以及切除的寡聚物的命运如何。在这里，我们证明体内的双切口与体外反应相同。此外，我们表明，在没有 XPC 蛋白的情况下进行的转录偶联修复也会在 UV 照射的 XP-C 突变细胞中产生名义上的 30-mer。最后，我们报告说，切除的 30-mer 与修复因子 TFIIH 和 XPG 一起从染色质中释放。总之，我们的结果表明了人类核苷酸切除修复体内和体外数据的一致性。
