Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair
Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair
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DOI：
10.1073/pnas.1306160110
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发表时间：
2013-10-15
影响因子：
11.1
通讯作者：
Kastan, Michael B.
中科院分区：
文献类型：
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作者：
Goldstein, Michael;Derheimer, Frederick A.;Kastan, Michael B.
DNA修复因子的募集和DNA双链断裂（DSB）位点染色质结构的调节是一个复杂且高度协调的过程。我们开发了一种系统，可以在哺乳动物基因组中确定的内源性位点快速诱导DSB，并能够直接评估修复并监测DSB的蛋白质募集、排出和修饰。系统的严格调节还允许评估与细胞对DNA断裂的反应相关的事件的相对动力学和依赖性。该系统的独特优势，超过焦点形成/消失测定评估DSB修复证明。使用ChIP，我们发现，核小体在G1期阻滞的哺乳动物细胞中的非同源末端连接修复过程中，在DSB周围部分解体，其特征在于H2 A/H2 B组蛋白二聚体的瞬时丢失。核仁素是一种具有组蛋白伴侣活性的蛋白质，通过其富含甘氨酸的结构域与RAD 50相互作用，并以MRE 11-NBS 1-RAD 50复合物依赖性方式快速募集到DSB。核仁素的下调消除了核小体破坏、修复因子的募集和DSB的修复，证明了核小体破坏在DSB修复中的功能重要性，并鉴定了该过程所需的染色质重塑蛋白。有趣的是，在循环细胞中DSB修复期间发生的核小体破坏的不同之处在于H2 A/H2 B和H3/H4组蛋白二聚体都被去除。这种完全的核小体破坏也依赖于核仁素，并且是将复制蛋白A募集到DSB所必需的，DSB是同源重组修复所必需的DSB加工的标志物。
Recruitment of DNA repair factors and modulation of chromatin structure at sites of DNA double-strand breaks (DSBs) is a complex and highly orchestrated process. We developed a system that can induce DSBs rapidly at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Distinct advantages of this system over focus formation/disappearance assays for assessing DSB repair are demonstrated. Using ChIP, we found that nucleosomes are partially disassembled around DSBs during nonhomologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its arginine-glycine rich domain and is recruited to DSBs rapidly in an MRE11-NBS1-RAD50 complex-dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and the repair of the DSB, demonstrating the functional importance of nucleosome disruption in DSB repair and identifying a chromatin-remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for recruitment of replication protein A to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair.