The Role of 53BP1 Interacting Proteins in Regulation of DNA Repair Choice

53BP1 相互作用蛋白在 DNA 修复选择调节中的作用

• 批准号: 8786396
• 负责人: Jinesh Shailesh Gheeya
• 金额: $ 3.32万
• 依托单位: RBHS -CANCER INSTITUTE OF NEW JERSEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2017-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786396
• 关键词: Animal Model; BRCA1 gene; Biological Assay; Breast; Candidate Disease Gene; Cell Cycle; Cell Cycle Checkpoint; Cell Line; Cells; Cisplatin; Complex; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA repair protein; Data; Defect; Excision; Functional disorder; Genomic Instability; Genomics; Health; Human; Immunohistochemistry; Inherited; Lead; Malignant Neoplasms; Malignant neoplasm of ovary; Measures; Mediating; Modality; Modeling; Mus; Mutation; Nonhomologous DNA End Joining; Nuclear Matrix-Associated Proteins; Ovarian; Pathway interactions; Phase; Phenotype; Play; Proteins; Proteomics; Recruitment Activity; Regulation; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; S Phase; Scaffolding Protein; Set protein; Site; Small Interfering RNA; Tumor Suppressor Proteins; Woman; base; cancer cell; crosslink; design; functional loss; high risk; homologous recombination; human H2AX protein; inhibitor/antagonist; innovation; insight; malignant breast neoplasm; mutant; novel; protein protein interaction; repaired; restoration

DESCRIPTION (provided by applicant): 53BP1 is a nuclear scaffolding protein and is involved in DNA repair and cell cycle checkpoint control. Upon induction of DNA double strand breaks, 53BP1 is rapidly phosphorylated by ATM/ATR, and recruited to sites of DNA breaks where it co-localizes with phosphorylated histone H2AX and other DNA repair proteins such as BRCA1. Women with functional loss of BRCA1 are at significantly higher risk for developing breast or ovarian malignancies. Cancer cells that are deficient in BRCA1 have significant homologous recombination mediated DNA repair (HDR) defect, genomic instability, and sensitivity to PARP inhibitors (PARPi) and cisplatin. The current model is that cells with BRCA1 deficiency have unconstrained 53BP1 activity. 53BP1, by yet uncharacterized mechanism, inhibits end resection at DNA double strand breaks and thus, promoting non- homologous end joining (NHEJ) and suppressing HDR. Interestingly, cells with both BRCA1 and 53BP1 deficiencies have partial restoration of HDR and resistance to PARPi and cisplatin. These observations have given rise to notion that 53BP1 and BRCA1 are critical regulators of DNA repair choice. The mechanism, however, by which 53BP1 executes its function in inhibiting end-resection and HDR, and promoting NHEJ is completely unknown. 53BP1 has no known enzymatic function, and is thought to function as a scaffold protein that acts as a platform for protein-protein interactions. We propose that 53BP1 interacts with a set of proteins that are necessary for its role in inhibiting end resection. These interactions are critical for the suppression of HDR seen in BRCA1-/- cells. Loss of 53BP1 interacting proteins will restore HDR in BRCA1 -/- cells and lead to chemoresistance. Recent studies have identified RAD50, a component of MRN complex, and TopBP1, a regulator of CtIP, as 53BP1 interacting proteins. As the MRN complex and CtIP plays critical role in end resection and HDR, it is plausible that 53BP1 mediates the DNA repair phenotype in Brca1 - /- cells via RAD50 and TopBP1. siRNA knockdown will be performed on 53BP1 interacting proteins in Brca1 -/- cell line to measure the restoration of HDR and resistance to PARPi and cisplatin. Furthermore, we will use unbiased high-throughput proteomics approach to identify novel 53BP1-interacting proteins in both Brca1 +/+ and Brca1-/- cell lines. Role of these candidate genes will be analyzed in DNA repair using specific GFP-based assays for HR and NHEJ. Expression of known and novel 53BP1 interacting proteins in human and murine BRAC1-related cancers will be determined using immunohistochemistry, RT-PCR, and genomic sequencing. The ultimate aim of this proposal is to gain new insight into how 53BP1 functions to regulate DNA repair choice in both BRCA1-mutant and BRCA1-wt cells. This will lead to new understanding of how DNA repair pathways are regulated as well as innovative approaches to target chemo resistance in human cancers.

描述（由申请人提供）：53 BP 1是一种核支架蛋白，参与DNA修复和细胞周期检查点控制。在诱导DNA双链断裂时，53 BP 1被ATM/ATR快速磷酸化，并被募集到DNA断裂位点，在那里它与磷酸化的组蛋白H2 AX和其他DNA修复蛋白如BRCA 1共定位。BRCA 1功能缺失的女性患乳腺癌或卵巢恶性肿瘤的风险明显更高。BRCA 1缺陷的癌细胞具有显著的同源重组介导的DNA修复（HDR）缺陷、基因组不稳定性以及对PARP抑制剂（PARPi）和顺铂的敏感性。目前的模型是BRCA 1缺陷的细胞具有不受约束的53 BP 1活性。53 BP 1通过尚未表征的机制抑制DNA双链断裂处的末端切除，从而促进非同源末端连接（NHEJ）并抑制HDR。有趣的是，具有BRCA 1和53 BP 1缺陷的细胞具有HDR的部分恢复和对PARPi和顺铂的抗性。这些观察结果引起了这样的概念，即53 BP 1和BRCA 1是DNA修复选择的关键调节因子。然而，53 BP 1在抑制末端切除和HDR以及促进NHEJ中执行其功能的机制完全未知。53 BP 1没有已知的酶功能，被认为是一种支架蛋白，作为蛋白质-蛋白质相互作用的平台。 我们提出，53 BP 1与一组蛋白质相互作用，这些蛋白质是其抑制末端切除作用所必需的。这些相互作用对于BRCA 1-/-细胞中观察到的HDR抑制至关重要。53 BP 1相互作用蛋白的缺失将恢复BRCA 1-/-细胞中的HDR并导致化学抗性。最近的研究发现，MRN复合物的一个组成部分RAD 50和CtIP的一个调节因子TopBP 1是53 BP 1相互作用的蛋白。由于MRN复合物和CtIP在末端切除和HDR中起关键作用，因此53 BP 1通过RAD 50和TopBP 1介导Brca 1- /-细胞中的DNA修复表型是合理的。将对Brca 1-/-细胞系中的53 BP 1相互作用蛋白进行siRNA敲低，以测量HDR的恢复以及对PARPi和顺铂的抗性。此外，我们将使用公正的高通量蛋白质组学方法，以确定新的53 BP 1相互作用的蛋白在Brca 1 +/+和Brca 1-/-细胞系。将使用HR和NHEJ的基于GFP的特异性测定分析这些候选基因在DNA修复中的作用。将使用免疫组织化学、RT-PCR和基因组测序来确定已知和新型53 BP 1相互作用蛋白在人类和小鼠BRAC 1相关癌症中的表达。该提案的最终目的是获得对53 BP 1如何在BRCA 1突变体和BRCA 1野生型细胞中调节DNA修复选择的新见解。这将导致对DNA修复途径如何调节的新理解，以及靶向人类癌症化疗耐药性的创新方法。