Maher R L, Marsden C G, Averill A M, Wallace S S, Sweasy J B, Pederson D S
Department of Microbiology and Molecular Genetics, and The Markey Center for Molecular Genetics, University of Vermont, Burlington, VT, 05405, USA.
Department of Microbiology and Molecular Genetics, and The Markey Center for Molecular Genetics, University of Vermont, Burlington, VT, 05405, USA; Departments of Therapeutic Radiology and Human Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.
DNA Repair (Amst). 2017 Sep;57:91-97. doi: 10.1016/j.dnarep.2017.06.029. Epub 2017 Jul 5.
Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg/ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg/ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes.
活性氧每天在每个人类细胞中产生约20,000个碱基损伤。这些潜在的诱变或细胞毒性损伤中的绝大多数都要经过碱基切除修复（BER）。尽管染色质重塑因子已被证明在体外可增强从核小体中切除氧化碱基的能力，但尚不清楚它们在体内是否会被招募到BER位点并在该位点发挥作用。为了验证细胞具有增强染色质中BER的因子这一假设，我们评估了人类细胞核提取物从外源添加的模型核小体中切除胸腺嘧啶乙二醇（Tg）损伤的能力。这些提取物中的DNA糖基化酶NTHL1能够从裸露的DNA以及早期研究表明空间可及的核小体位点切除Tg。然而，只有在添加Mg/ATP后，相同的提取物才能从核小体中空间受阻的位点切除损伤。凝胶迁移率变动分析表明，在Mg/ATP依赖性切除反应后，核小体基本保持完整。尺寸排阻色谱表明，NTHL1刺激活性的分子量相对较低，接近NTHL1和其他BER糖基化酶；含有非常大的染色质重塑复合物的柱级分没有表现出相同的刺激活性。这些结果表明，细胞具有促进染色质中BER起始的一种或多种因子，但与大多数已知的染色质重塑复合物不同。
