Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS.
Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS.
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DOI：
10.1186/1756-0500-4-13
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发表时间：
2011-01-21
影响因子：
1.8
通讯作者：
De Benedetti A
中科院分区：
文献类型：
--
作者：
Kanikarla-Marie P;Ronald S;De Benedetti A
利用 HO 核酸内切酶进行双链断裂 (DSB) 修复的酿酒酵母交配型开关模型是同源重组修复 (HRR) 和直接末端连接修复（非同源末端连接 - NHEJ）研究得最好的系统之一。我们最近利用表达 HO 的腺病毒和整合的基因组靶标将该系统转移到哺乳动物细胞培养模型中。这使得直接比较酵母细胞和哺乳动物细胞之间相同类型的诱导 DSB 的修复机制成为可能。 DSB 修复的研究强调了生物体之间特征、蛋白质和机制的共性，以及未发现保守性时的差异。酵母和哺乳动物细胞之间有两种明显不同的蛋白质：DNA-PK（一种因 DSB 存在而激活的蛋白激酶）和 Artemis（一种核酸酶，其活性受 DNA-PK 和 ATM 调节）。在本报告中，我们描述了这两种蛋白质如何参与 DSB 末端加工的特定模式，特别是在异染色质背景下。我们之前发表过，H2O2 诱导的 DSB 的修复通常是准确的，并且通过简单地重新连接 H2O2 产生的内聚 3'-突出端来进行。在没有嘌呤霉素选择的情况下，这些细胞连续传代期间，基因座似乎变得更加异染色质并且通过显示多种特征而沉默。 1) 该位点变得更不易被 H2O 核酸内切酶切割； 2) 赋予嘌呤霉素抗性的puro mRNA的表达降低； 3) 该位点的核小体占有率（组蛋白 H3 的 ChIP）增加，这是染色质更浓缩的指标。通过添加嘌呤霉素重新选择这些细胞后，许多这些特征被逆转。然而，即使是重新选择的细胞，其裂解和修复模式也与最初创建时的细胞不同。具体来说，修复模式揭示了 DSB 处的离散缺失，表明重新连接前核小体（或其他蛋白质复合物）发生单位损失，以 PCR 产物阶梯表示，让人想起细胞凋亡期间通常观察到的核小体间裂解。这种切割模式向我们表明，也许 Artemis（一种被认为在细胞凋亡和 DSB 修复过程中产生核小体片段的蛋白质）参与了末端加工的特定模式。初步证据表明情况可能如此，因为用 siRNA 敲低 Artemis 消除了阶梯模式，并揭示了重新连接前末端的广泛核酸外切加工。我们在哺乳动物细胞中构建了一个系统，其中正选择的缺失导致目标位点处的染色质重塑，这概括了酵母中交配型转换系统的许多特征。具体来说，正如酵母 HML 和 HMR 一样，该位点的转录受到抑制； HO 核酸内切酶切割的可及性降低；末端的处理也发生了巨大的变化。这种转变是从粘性末端的高保真再连接转变为核小体间片段的释放模式，可能是为了寻找用于连接的微同源性延伸。这与报道一致，即 ATM、DNA-PK 和 Artemis 参与 DSB 修复主要集中在异染色质区域，而常染色质中大多数 IR 诱导的 DSB 修复灶并不需要。
The S. cerevisiae mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin. We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation. e have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.