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

Ku70 结合蛋白-5 (KUB5)，非同源末端连接的新因子

• 批准号: 8017553
• 负责人: David A Boothman
• 金额: $ 32.89万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8017553
• 关键词: 5' Untranslated Regions; ATM activation; Affect; Binding; Binding Proteins; Biochemical; Breast; Breast Cancer Cell; C-terminal; Cell Line; Cell physiology; Cells; Chromatids; Chromatin Loop; Cisplatin; Coiled-Coil Domain; Comet Assay; Complementary DNA; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA repair protein; DNA-Directed RNA Polymerase; Data; Defect; Depressed mood; Development; Diploidy; Double Strand Break Repair; Embryo; Exonuclease; Failure; Fatty acid glycerol esters; Fibroblasts; Future; G22P1 gene; Gel Chromatography; Genes; Genetic; Genetic Transcription; Genomic Instability; Genomics; Growth; Half-Life; Haploidy; Heterozygote; Homologous Gene; Human; Hybrids; Hypersensitivity; Ionizing radiation; Knock-out; Knockout Mice; Ligation; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Mediating; Modeling; Monitor; Mus; N-terminal; Nature; Nonhomologous DNA End Joining; Nuclear Receptor Coactivator 3; Pharmaceutical Preparations; Phase; Phenotype; Play; Polymerase; Process; Proteins; RNA; RNA Polymerase II; Radio; Regulation; Reporting; Resistance; Ribonuclease H; Role; Serine; Site; Small Interfering RNA; Source; Structure; Testing; Transcript; Untranslated Regions; Western Blotting; Yeasts; artemis; cancer cell; chemotherapy; homologous recombination; in vivo; killings; malignant breast neoplasm; mutant; novel; prevent; protein function; public health relevance; radiation resistance; repaired; resistance mechanism; response; small hairpin RNA; transcription termination; vector; yeast two hybrid system

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.

描述（由申请人提供）：DNA修复的改变在乳腺癌对电离辐射（IR）和化疗的抗性中起主要作用。在DSB修复和RNA转录中起作用的因子，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停止或转录不能正常终止时，RNA/DNA杂交体（r -环）的半衰期延长，导致DNA双链断裂（DSB）的形成和遗传不稳定。通过酵母双杂交分析，我们鉴定了Ku70结合蛋白#5 [(Kub5)/Hera]，这是酵母rtt103的一个高度保守的人类同源物，通过调控RNA Pol II来终止RNA转录。无论是通过敲除杂合子还是通过siRNA/shRNA表达敲低Kub5/Hera表达，Kub5/Hera表达的稳定缺失都会导致基础dsb升高、ATM激活、病灶形成和染色单体畸变，而这些都被RNase H强制过表达所消除，这表明r环在遗传不稳定中发挥了作用。Kub5/Hera敲低细胞对IR过敏，相当于Ku70缺陷细胞，不能修复需要DNA末端加工的dsb。我们假设KUB5/HERA在细胞中发挥双重作用：(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相互作用刺激复杂dsb的NHEJ加工。当其表达被抑制时（例如，由于单倍体不足而产生一半），r -环形成并由于RNA转录终止缺陷而产生dsb。DNA损伤同时被细胞无法修复下游的dsb放大，导致IR超敏反应。目标1：对Kub5/Hera的两个区域（RPR和coil -coil结构域）进行结构/功能分析，解耦转录终止和DSB修复；目的2：明确KUB5- Ku70-Artemis复合物在DSB修复中的功能；目的3：确定KUB5/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KUB5/HERA过表达在人乳腺癌放射耐药中的作用。