Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):16874-9. doi: 10.1073/pnas.1306160110. Epub 2013 Sep 30.
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.
DNA 修复因子的募集和 DNA 双链断裂 (DSB) 部位染色质结构的调节是一个复杂而高度协调的过程。我们开发了一种系统，可以在哺乳动物基因组中的内源性特定位置快速诱导 DSB，并能够直接评估修复，监测 DSB 处的蛋白质募集、流出和修饰。该系统的严格调控还允许评估与细胞对 DNA 断裂反应相关的事件的相对动力学和依赖性。与用于评估 DSB 修复的焦点形成/消失测定相比，该系统具有明显的优势。通过 ChIP，我们发现，在 G1 期停滞的哺乳动物细胞中非同源末端连接修复过程中，DSB 周围的核小体部分解聚，其特征是 H2A/H2B 组蛋白二聚体的短暂丢失。核仁素是一种具有组蛋白伴侣活性的蛋白质，通过其精氨酸-甘氨酸丰富结构域与 RAD50 相互作用，并通过 MRE11-NBS1-RAD50 复合物依赖性方式快速募集到 DSB 。核仁素的下调会破坏核小体的破坏、修复因子的募集以及 DSB 的修复，这表明核小体破坏在 DSB 修复中的功能重要性，并确定了该过程所需的染色质重塑蛋白。有趣的是，在有丝分裂细胞中发生的 DSB 修复过程中的核小体破坏不同，因为 H2A/H2B 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，并且对于复制蛋白 A 募集到 DSB 是必需的，这是同源重组修复的必需标志物。
