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Determining the effects of RAD51C ovarian cancer variants

确定 RAD51C 卵巢癌变异的影响

基本信息

批准号：
10461516
负责人：
Hayley Rein
金额：
$ 4.68万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2024-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10461516
关键词：
ATP phosphohydrolase Affect Biological Assay CRISPR/Cas technology Cells Cisplatin Comet Assay Complement Complex DNA DNA Damage DNA Double Strand Break DNA Repair Data Defect Double Strand Break Repair Fiber Filament Fluorescence Microscopy Gene Duplication Hereditary Breast Carcinoma Human Hybrids Individual Initiator Codon Kinetics Knock-out Lead Malignant Neoplasms Malignant neoplasm of ovary Mediating Microscopy Molecular Mutate Mutation Nuclear Extract Ovarian Pathway interactions Patients Persons Point Mutation Process Production Proteasome Inhibitor Protein Isoforms Proteins RAD51C gene Rad51 recombinase Regulation Resected Risk Serous Site Time Translations Variant Western Blotting Work Yeasts cancer cell cancer prevention cancer subtypes fluorescence microscope gamma irradiation genetic variant homologous recombination irradiation mutant paralogous gene prevent protein degradation repaired ribosome profiling single molecule treatment planning treatment response variant of unknown significance

项目摘要

Project Summary/ Abstract Approximately 50% of high grade serous ovarian cancers (HGSOC) are deficient in homologous recombination (HR). HR is a high-fidelity DNA double-strand break repair pathway which uses a homologous template for accurate DNA repair. The central protein of this process, RAD51, is tightly regulated. One group of proteins that work to regulate RAD51 are the RAD51 paralogs, including RAD51C and RAD51D. Collectively, RAD51C and RAD51D are mutated in 8% of HR deficient familial ovarian cancers. Although the RAD51 paralogs were discovered 20+ years ago their exact function in HR, and how mutation in these proteins contribute to HGSOC is still unknown. We have identified two possible RAD51C protein isoforms which may perform separate function in HR. RAD51C variants that would prevent protein production of either isoform have been identified in 32 separate individuals. Thus, we propose a functional analysis of these isoforms and mutations which disrupt their production. In addition, we have also identified mutations in RAD51C which disrupt its interaction with RAD51D. Our preliminary data has determined that the yeast-three-hybrid interaction between RAD51C and RAD51D is a good indicator of HR efficiency in human cells. Yet, how this RAD51C-RAD51D interaction contributes to HR is still unknown. We will examine the effects of these variants on RAD51 protein regulation and filament dynamics using single-molecule fluorescence microscopy (C-trap). By characterizing the activity of these RAD51C variants we will uncover both the function of WT-RAD51C and how dysfunctional RAD51C variants contribute to HR deficiency in HGSOC.

项目总结/摘要大约50%的高级别浆液性卵巢癌（HGSOC）缺乏同源重组（人力资源）。HR是一种高保真DNA双链断裂修复途径，其使用同源模板来修复DNA双链断裂。精确的DNA修复这一过程的中心蛋白质RAD 51受到严格调控。一组蛋白质调节RAD 51的是RAD 51旁系同源物，包括RAD 51 C和RAD 51 D。RAD51C 和RAD 51 D在8%的HR缺陷型家族性卵巢癌中突变。尽管RAD 51旁系同源物 20多年前发现了它们在HR中的确切功能，以及这些蛋白质的突变如何促成HGSOC 仍然未知。我们已经鉴定了两种可能的RAD 51 C蛋白同种型，它们可以单独地执行功能。 RAD 51 C变体中的功能，其将阻止任一同种型的蛋白质产生。 32个独立的个体因此，我们提出了这些异构体和突变的功能分析，破坏，他们的生产。此外，我们还鉴定了RAD 51 C中的突变，这些突变破坏了RAD 51 C与 RAD51D。我们的初步数据已经确定，RAD 51 C和 RAD 51 D是人类细胞中HR效率的良好指标。然而，这种RAD 51 C-RAD 51 D相互作用对HR的贡献是未知的。我们将研究这些变体对RAD 51蛋白调节的影响。和使用单分子荧光显微镜（C-trap）的细丝动力学。通过描述在这些RAD 51 C变体中，我们将揭示WT-RAD 51 C的功能以及功能失调的RAD 51 C是如何产生的。变异导致HGSOC中的HR缺陷。