The exonuclease activity of hPMC2 is required for transcriptional regulation of the QR gene and repair of estrogen-induced abasic sites.
The exonuclease activity of hPMC2 is required for transcriptional regulation of the QR gene and repair of estrogen-induced abasic sites.
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DOI：
10.1038/onc.2011.186
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发表时间：
2011-11-24
期刊：
影响因子：
8
通讯作者：
Montano, M. M.
中科院分区：
文献类型：
--
作者：
Krishnamurthy, N.;Ngam, C. R.;Berdis, A. J.;Montano, M. M.
我们以前曾报道，反式羟基他莫昔芬（TOT）在乳腺上皮细胞系提供保护，对雌激素诱导的DNA损伤的抗氧化应激酶的表达上调。这种调节涉及雌激素受体β（ERβ）募集到亲电反应元件（EpRE）和一种新的蛋白质，即防止有丝分裂灾难的爪蟾基因（hPMC 2）的人类同源物。我们还证明了ERβ和hPMC 2是聚（ADP-核糖）聚合酶1（PARP-1）和拓扑异构酶IIβ（Topo IIβ）向EpRE的TOT依赖性募集所必需的。序列分析表明，hPMC 2的C-末端编码一个推定的核酸外切酶结构域。使用体外动力学测定，我们发现hPMC 2是降解单链和双链底物的3 '-5'非加工性核酸外切酶。两个保守的羧酸残基的突变大大降低了hPMC 2的核酸外切酶活性，表明催化残基的相对重要性。乳腺癌细胞系的醌还原酶（QR）水平的蛋白质印迹分析显示，TOT诱导的QR上调所需的hPMC 2的内在核酸外切酶活性。染色质免疫沉淀试验（ChIP）也表明，hPMC 2参与形成与TOT-治疗观察到的链断裂，是特异性的QR基因的含EpRE的区域。我们还确定了转录因子NF-E2相关因子-2（Nrf 2）参与hPMC 2对EpRE的特异性。此外，我们确定了hPMC 2的催化活性是修复由雌激素诱导的DNA损伤引起的脱碱基位点所必需的。因此，我们的研究为hPMC 2的转录调控提供了一个机制基础，并为其在癌症预防中的作用提供了新的见解。
We have previously reported that the expression of antioxidative stress enzymes are upregulated by trans-hydroxytamoxifen (TOT) in breast epithelial cell lines providing protection against estrogen-induced DNA damage. This regulation involves Estrogen Receptor beta (ERβ) recruitment to the Electrophile Response Element (EpRE) and a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). We have also demonstrated that ERβ and hPMC2 are required for TOT-dependent recruitment of poly (ADP-ribose) polymerase 1 (PARP-1) and Topoisomerase IIβ (Topo IIβ) to the EpRE. Sequence analysis reveals that the C-terminus of hPMC2 encodes a putative exonuclease domain. Using in vitro kinetic assays, we found that hPMC2 is a 3'–5' non-processive exonuclease that degrades both single stranded and double stranded substrates. Mutation of two conserved carboxylate residues drastically reduced the exonuclease activity of hPMC2 indicating the relative importance of the catalytic residues. Western blot analysis of breast cancer cell lines for Quinone Reductase (QR) levels revealed that the intrinsic exonuclease activity of hPMC2 was required for TOT-induced QR upregulation. Chromatin immunoprecipitation assays (ChIP) also indicated that hPMC2 was involved in the formation of strand breaks observed with TOT-treatment and is specific for the EpRE-containing region of the QR gene. We also determined that the transcription factor NF-E2-related factor-2 (Nrf2) is involved in the specificity of hPMC2 for the EpRE. In addition, we determined that the catalytic activity of hPMC2 is required for repair of abasic sites that result from estrogen-induced DNA damage. Thus our study provides a mechanistic basis for transcriptional regulation by hPMC2 and provides novel insights into its role in cancer prevention.
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