Study of the DNA damage checkpoint using Xenopus egg extracts.
Study of the DNA damage checkpoint using Xenopus egg extracts.
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DOI：
10.3791/4449
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发表时间：
2012-11
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影响因子：
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通讯作者：
Jeremy Willis;D. Destephanis;Y. Patel;Vrushab Gowda;Shan Yan
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作者：
Jeremy Willis;D. Destephanis;Y. Patel;Vrushab Gowda;Shan Yan
每天，细胞都会受到各种内源性和环境的损伤。为了对抗这些损伤，细胞已经进化出DNA损伤检查点信号传导作为一种监测机制，以感知DNA损伤并指导细胞对DNA损伤的反应。有几组蛋白质称为传感器，换能器和效应器参与DNA损伤检查点信号传导（图1）。在这个复杂的信号通路中，ATR（ATM and Rad 3-related）是能够响应DNA损伤和复制应激的主要激酶之一。激活的ATR可以磷酸化其下游底物，如Chk 1（检查点激酶1）。因此，磷酸化和激活的Chk 1导致DNA损伤检查点中的许多下游效应，包括细胞周期停滞、转录激活、DNA损伤修复和凋亡或衰老（图1）。当DNA受损时，未能激活DNA损伤检查点会导致未修复的损伤，随后导致基因组不稳定。对DNA损伤检查点的研究将阐明细胞如何保持基因组完整性，并更好地了解人类疾病（如癌症）如何发展。非洲爪蟾卵提取物是DNA损伤检查点研究中一种有效的无细胞提取物模型系统。低速提取（LSE）最初由Masui小组描述。将脱膜精子染色质添加到LSE中导致细胞核形成，其中DNA在每个细胞周期中以保守方式复制一次。ATR/Chk 1介导的检查点信号通路由DNA损伤或复制应激触发。目前，诱导DNA损伤检查点的方法主要有两种：DNA损伤方法和DNA损伤模拟结构。紫外线、γ射线、甲基磺酸甲酯、丝裂霉素C、4-硝基喹啉氧化物、阿非迪霉素等均可引起DNA损伤。MMS是抑制DNA复制并激活ATR/Chk 1介导的DNA损伤检查点的烷化剂。紫外线照射还触发ATR/Chk 1依赖的DNA损伤检查点。DNA损伤模拟结构AT 70是两个寡核苷酸poly-（dA）70和poly-（dT）70的退火复合物。AT 70系统是在Bill Dunphy的实验室开发的，广泛用于诱导ATR/Chk 1检查点信号。在这里，我们描述了（1）制备无细胞卵提取物（LSE），（2）用两种不同的DNA损伤方法（MMS和UV）处理非洲爪蟾精子染色质，（3）制备DNA损伤模拟结构AT 70，以及（4）用受损的精子染色质或DNA损伤模拟结构在LSE中触发ATR/Chk 1介导的DNA损伤检查点的方案。
On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.