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

描述(申请人提供)：DNA修复的改变在乳腺癌对电离辐射(IR)和化疗的抵抗中发挥着重要作用。影响DSB修复和RNA转录的因素，特别是调节RNA聚合酶(Pol)II的蛋白质，已经被提出，但很少有报道。当RNA Pol II停滞或转录不能正确终止时，RNA/DNA杂交物(R环)的半衰期延长，导致DNA双链断裂(DSB)形成和遗传不稳定。利用酵母双杂交分析，我们鉴定了Ku70结合蛋白#5[(Kub5)/Hera]，它是酵母rtt103的一个高度保守的人类同源物，通过其对RNA Pol II的调控终止RNA转录。Kub5/Hera表达的稳定丢失，无论是通过杂合子敲除还是通过siRNA/shRNA表达下调，都会导致基础DSB增加，ATM激活，焦点形成和染色单体畸变被RNaseH强制过表达所消除，这表明R-环在遗传不稳定性中发挥了作用。Kub5/Hera基因敲除细胞对IR高度敏感，相当于Ku70缺陷细胞，不能修复需要DNA末端处理的DSB。我们假设KUB5/HERA在细胞中起双重作用：(I)通过与RNA Pol II CTD结构域的相互作用调节RNA Pol II终止RNA转录；(Ii)通过Ku70和Artemis的相互作用刺激复杂DSB的NHEJ加工。当它的表达被抑制时(例如，一半是由于单链不足)，R-环形成，由于RNA转录终止缺陷而产生DSB。DNA损伤同时由于细胞无法修复下游的DSB而放大，导致IR过敏症。目标1：对KUB5/HERA的结构和功能进行分析，主要针对RPR和螺旋线圈结构域，以了解转录终止和DSB修复；目标2：确定KUB5-Ku70-Artemis复合体在DSB修复中的功能；以及目标3：确定KUB5/HERA在调节RNAPOL II功能、RNA转录终止、自发DSB形成和染色单体异常中的作用(S)。这些研究将使我们能够探索KUB5/HERA过度表达在人类乳腺癌放射耐药中的作用。 与公共卫生相关：人类Ku70结合蛋白#5，也称为HERA，(KUB5/HERA)是一种新的因子，参与修复具有复杂损伤的DNA末端，如钝端断裂。KUB5/HERA在乳腺癌和卵巢癌中的升高，以及我们的证据表明，KUB5/HERA对药物和辐射耐药极其重要，这表明该蛋白是未来治疗的有价值的靶点，并是治疗反应的“有价值的预测指标”。我们最近观察到KUB5/HERA是RNA终止所必需的，这使我们能够研究DNA修复与RNA聚合酶II调控和转录终止之间的接口。

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.