Ku70 Binding Protein-5 (KUB5), a Novel Factor in Nonhomologous End Joining

描述（由申请人提供）：DNA修复的改变在乳腺癌对电离辐射（IR）和化疗的耐药性中起主要作用。在DSB修复和RNA转录中起作用的因素，特别是调节RNA聚合酶（Pol）II的蛋白质，已经被提出，但很少报道。当RNA Pol II停滞时，或者当转录不能正确终止时，RNA/DNA杂合体（R环）具有延长的半衰期，导致DNA双链断裂（DSB）形成和遗传不稳定性。 使用酵母双杂交分析，我们鉴定了Ku 70结合蛋白#5 [（Kub 5）/Hera]，这是酵母rtt 103的高度保守的人类同源物，其通过其对RNA Pol II的调节终止RNA转录。通过杂合子敲除或通过siRNA/shRNA表达敲减的Kub 5/Hera表达的稳定丧失导致升高的基础DSB、ATM活化、病灶形成和染色单体畸变，其被RNase H强制过表达消除，表明R环在遗传不稳定性中的作用。Kub 5/Hera敲除细胞对IR高度敏感，相当于Ku 70缺陷细胞，并且不能修复需要DNA末端加工的DSB。 我们假设KUB 5/HERA在细胞中发挥双重作用：（i）通过其与RNA Pol II CTD结构域的相互作用调节RNA Pol II以终止RNA转录;以及（ii）通过Ku 70和Artemis相互作用刺激NHEJ加工复杂的DSB。当其表达被抑制时（例如，一半通过单倍体不足）形成R环，并且由于缺陷的RNA转录终止而产生DSB。DNA损伤同时被细胞无法修复下游DSB放大，导致IR超敏反应。提出了三个具体目的：目的1：进行Kub 5/Hera的结构/功能分析，重点是RPR和卷曲螺旋结构域两个区域，以解偶联转录终止和DSB修复;目的2：确定Kub 5-Ku 70-Artemis复合物在DSB修复中的功能;目的3：确定KUB 5/HERA在调节RNA Pol II功能、RNA转录终止、自发DSB形成和染色单体畸变中的作用。这些研究将使我们能够探索KUB 5/HERA过表达在人类乳腺癌放射抵抗中的作用。 公共卫生相关性：人Ku 70结合蛋白#5，也称为HERA，（KUB 5/HERA）是一种新的参与修复具有复杂损伤的DNA末端的因子，例如平端断裂。 乳腺癌和卵巢癌中KUB 5/HERA的升高，以及我们证明KUB 5/HERA对药物和辐射抗性极其重要，表明这种蛋白质是未来治疗的有价值的靶点，也是治疗的“有价值的反应预测因子”。我们最近观察到KUB 5/HERA是RNA终止所必需的，这使我们能够研究DNA修复和RNA聚合酶II调控与转录终止之间的界面。

DESCRIPTION (provided by applicant): Alterations in DNA repair play major roles in breast cancer resistance to ionizing radiation (IR) and chemotherapies. Factors operating in both DSB repair and RNA transcription, specifically proteins regulating RNA polymerase (Pol) II, have been suggested, but few reported. When RNA Pol II stalls, or when transcription cannot properly terminate, RNA/DNA hybrids (R-loops) have extended half-lives that result in DNA double strand break (DSB) formation and genetic instability. Using yeast two hybrid analyses, we identified Ku70 binding protein #5 [(Kub5)/Hera], a highly conserved human homolog of yeast rtt103 that terminates RNA transcription via its regulation of RNA Pol II. Stable loss of Kub5/Hera expression, either by heterozygote knockout or knockdown by siRNA/shRNA expression, leads to elevated basal DSBs, ATM activation, foci formation and chromatid aberrations that were abrogated by RNase H forced over- expression, suggesting a role for R-loops in genetic instability. Kub5/Hera knockdown cells were hypersensitive to IR, equivalent to Ku70 deficient cells, and failed to repair DSBs that require DNA end-processing. We hypothesize that KUB5/HERA plays dual roles in the cell to: (i) regulate RNA Pol II to terminate RNA transcription via its interaction with the RNA Pol II CTD domain; and (ii) stimulate NHEJ processing of complex DSBs by Ku70 and Artemis interactions. When its expression is depressed (e.g., one-half via haplo-insufficiency) R-loops form and DSBs are created due to deficient RNA transcription termination. DNA damage is simultaneously amplified by the cell's inability to repair DSBs downstream, leading to IR hypersensitivity. Three Specific Aims are proposed: Aim 1: to perform structure/function analyses of Kub5/Hera focusing on two regions, the RPR and coiled-coil domains, to uncouple transcription termination and DSB repair; Aim 2: to define functions of KUB5- Ku70-Artemis complexes in DSB repair; and Aim 3: to define the role(s) of KUB5/HERA in regulating RNA Pol II function, RNA transcription termination, and spontaneous DSB formation and chromatid aberrations. These studies will allow us to explore the role of KUB5/HERA over-expression in human breast cancer radio-resistance. PUBLIC HEALTH RELEVANCE: Human Ku70 binding protein #5, also known as HERA, (KUB5/HERA) is a new factor involved in the repair of DNA ends with complex damage, such as blunt-end breaks. Elevations of KUB5/HERA in breast and ovarian cancers, and our demonstration that KUB5/HERA is extremely important for drug and radiation resistance, suggests that this protein is a valuable target for future therapies and a 'valuable predictor of response' for therapies. Our recent observation that KUB5/HERA is required for RNA termination allows us to also investigate the interface between DNA repair and regulation of RNA Polymerase II and transcription termination.