Regulation of Ionizing Radiation-Induced DNA Damage Response

描述（由申请人提供）： 电离辐射（IR）是通过诱导DNA双链断裂（DSB）导致细胞凋亡来杀伤肿瘤细胞的重要治疗手段。然而，正常细胞通过DNA损伤应答系统（包括DNA损伤检查点激活和DNA损伤修复）保护免于IR诱导的细胞致死。因此，了解辐射诱导的DNA损伤反应的分子机制，从而实现更有效的放射治疗癌症患者是非常重要的。 在IR诱导的DSB反应中，由一组PI3样激酶（包括ATM、ATR和DNAPK）启动的信号级联反应阻止细胞周期进展并促进DNA损伤修复。除了这些蛋白质磷酸化事件，我们和其他人最近发现，蛋白质泛素化级联反应参与DSB反应。这些泛素化事件被RNF8激活，RNF8是一种环结构域E3连接酶。在最初的RNF8依赖性泛素化之后，泛素（ub）信号被一组下游ub E3连接酶（如RNF168、RNF169、RAD 18和HERC2）放大。这些泛素化事件调节染色质重塑和其他组蛋白标记，这通过将下游DNA损伤修复因子募集到DSB来促进DNA损伤修复。 在本申请中，我们计划继续研究IR诱导的蛋白质泛素化级联反应，重点是CHFR，哺乳动物中RNF8的一个parasites。CHFR和RNF8的结构域结构非常相似。它们都含有一个可能识别磷酸化Thr的N端FHA结构域和一个与Ubc13或UbcH5C相互作用以催化组蛋白泛素化的Ring结构域。缺乏这两种E3 ub连接酶会叠加抑制DNA损伤反应并诱导体内肿瘤发生。与RNF8不同的是，CHFR含有一个C-末端PBZ基序，该基序结合聚（ADP-核糖）（PAR），这有助于CHFR快速募集到DNA损伤位点并介导PARP1的泛素化。此外，CHFR缺陷小鼠易患肿瘤，并且已在原发性实体瘤中鉴定出癌症相关的CHFR基因突变。这些证据表明CHFR是一种重要的肿瘤抑制因子。因此，在本申请中，我们计划研究CHFR在IR诱导的DNA损伤反应和肿瘤抑制中的分子机制。

DESCRIPTION (provided by applicant): Ionizing radiation (IR) is an important therapeutic approach to kill cancer cells by inducing DNA double strand breaks (DSBs) that lead to cell apoptosis. However, normal cells are protected from IR-induced cell lethality by a DNA damage response system including DNA damage checkpoint activation and DNA damage repair. Thus, it is important for understanding the molecular mechanism of IR-induced DNA damage response, so that more effective radiation therapy can be achieved to treat cancer patients. In response to IR-induced DSBs, a signal cascade initiated by a group of PI3-like kinases including ATM, ATR and DNAPK arrests cell cycle progression and facilitates DNA damage repair. Besides these protein phosphorylation events, we and others recently found that a protein ubiquitination cascade is involved in DSBs response. These ubiquitination events are activated by RNF8, a Ring domain E3 ligase. Following the initial RNF8-dependent ubiquitination, the ubiquitin (ub) signals are amplified by a group of downstream ub E3 ligases, such as RNF168, RNF169, RAD18, and HERC2. These ubiquitination events regulate chromatin remodeling and other histone marks, which facilitates DNA damage repair by recruiting down-stream DNA damage repair factors to DSBs. In this application, we plan to continue studying the IR-induced protein ubiquitination cascade by focusing on CHFR, a paralog of RNF8 in mammals. The domain architecture of CHFR and RNF8 is very similar. Both of them contain an N-terminal FHA domain that is likely to recognize phospho-Thr, and a Ring domain that interacts Ubc13 or UbcH5C to catalyze histone ubiquitination in response to DSBs. Lacking these two E3 ub ligases additively suppress DNA damage response and induces tumorigenesis in vivo. Different from RNF8, CHFR contains a C-terminal PBZ motif that binds poly(ADP-ribose) (PAR), which facilitates the fast recruitment of CHFR to DNA damage sites and mediates the ubiquitinaition of PARP1. Moreover, Chfr-deficient mice are tumor prone, and cancer-associated CHFR gene mutations have been identified in primary solid tumors. These lines of evidence suggest that CHFR is an important tumor suppressor. Thus, in this application, we plan to examine the molecular mechanism of CHFR in IR-induced DNA damage response and tumor suppression.