Chromatin's Role in Repair of Radiation-induced Damage

本研究的目的是确定染色质在辐射诱导的双链断裂（DSBs）修复过程中所起的基本作用。在哺乳动物细胞中，细胞存活和基因组完整性的维持主要依赖于非同源末端连接（NHEJ）途径对DSBs的修复。如果修复不正确，dsb会导致畸变，如染色体重排，并可能导致癌症的形成。为了充分了解辐射诱导的dsb修复，考虑自然环境-染色质是很重要的。基因组包装成染色质可能会影响DNA修复过程，类似于基因表达的情况。因此，我们发现了一种新的染色质组装因子，称为ASF1，它对体内辐射诱导的内源性和发育程序性DNA损伤的修复至关重要。令人惊讶的是，ASF1甚至是DNA修复过程（如NHEJ）所必需的，而这些修复过程并不调用染色质组装到新合成的DNA上。我们将测试在NHEJ过程中染色质结构被ASF1染色质组装因子改变的假设，并且染色质结构的改变对于DNA损伤的修复是必不可少的和内在的。为了深入了解辐射诱导DNA损伤的修复，该方法将是在酵母内诱导一种高度特异性的核酸内切酶，以产生一种只能由NHEJ修复的同步定义的DSB。利用这个模型系统，我们将首次绘制出NHEJ修复DSB之前、伴随和之后的染色质结构变化。最后，我们将通过检查ASF1和染色质结构在哺乳动物细胞中的作用来确定染色质结构对NHEJ影响的普遍性。使用模型遗传系统诱导确定的DSB的实验结果将为理解辐射诱导的DSB修复过程中染色质结构的基本作用（但以前被忽视）提供基础。因此，这些研究直接适用于因辐射引起的基因组完整性丧失而导致的人类疾病，包括许多形式的癌症。

The goal of this research is to define the fundamental role that chromatin plays during the repair of radiation-induced double-strand breaks (DSBs). Cell survival and maintenance of genome integrity are critically dependent on the repair of DSBs by the non-homologous end joining (NHEJ) pathway in mammalian cells. If repaired incorrectly, DSBs result in abberations such as chromosomal rearrangements and can lead to formation of cancers. In order to fully understand the repair of radiation- induced DSBs, it is important to consider the natural context - chromatin. The packaging of the genome into chromatin is likely to influence DNA repair processes by analogy to the situation with gene expression. Accordingly, we have discovered a novel chromatin assembly factor, termed ASF1, essential for the repair of radiation-induced, endogenous and developmentally-programmed DNA damage in vivo. Surprisingly, ASF1 is even required for DNA repair processes, such as NHEJ, that do not invoke the assembly of chromatin onto newly-synthesized DNA. We will test the hypothesis that chromatin structure is altered by the ASF1 chromatin assembly factor during NHEJ and that changes to the chromatin structure are essential and intrinsic to the repair of DNA damage. In order to gain insight into the repair of radiation-induced DNA damage, the approach will be to induce a highly specific endonuclease within yeast to generate a synchronous defined DSB that can only be repaired by NHEJ. Using this model system, we will map for the first time the changes to the chromatin structure that precede, accompany and follow the repair of a DSB by NHEJ. Finally, we will determine the generality of the influence of chromatin structure on NHEJ, by examining the role of ASF1 and chromatin structure in mammalian cells. The findings of the proposed experiments using model genetic systems to induce a defined DSB will provide the foundation for understanding the fundamental, yet previously overlooked, role of chromatin structure during the repair of radiation-induced DSBs. As such, these studies are directly applicable to human diseases that result from radiation-induced loss of genome integrity, including many forms of cancer.