FANCD2 Monoubiquitination in DNA Damage Responses

DNA 损伤反应中的 FANCD2 单泛素化

• 批准号: 7837429
• 负责人: PAUL R ANDREASSEN
• 金额: $ 23.31万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7837429
• 关键词: Acute Myelocytic Leukemia; Address; Apoptosis; CD34 gene; Cell physiology; Cells; Chromosomal Instability; Chromosome abnormality; Complex; Coupled; Crosslinker; DNA; DNA Damage; DNA Repair; DNA biosynthesis; Defect; Detection; Disease; Dysmyelopoietic Syndromes; Fanconi anemia protein; Fanconi' s Anemia; General Population; Genes; Goals; Hematopoiesis; Hematopoietic; Human; Hypersensitivity; Lead; Light; Link; MCM7 gene; Malignant Neoplasms; Mediating; Microscopy; Mitomycins; Molecular; Monoubiquitination; Mus; Mutate; Myelogenous; Normal Cell; Nuclear; Pancytopenia; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Predisposition; Prevalence; Proteins; Recruitment Activity; Regulation; Resistance; Role; Site; Stem cells; Stress; Study Section; System; Time; abstracting; base; clinical phenotype; insight; leukemia; mutant; prevent; progenitor; protein function; reconstitution; response; sensor; small hairpin RNA; stem

Abstract Fanconi Anemia (FA) is a multi-genic disorder that results in progressive bone marrow failure and a strong predisposition to myelodysplastic syndrome and leukemia. FA proteins are involved in the cellular response to DNA damage by a largely unknown mechanism. A nuclear complex of at least 8 FA proteins is required for the monoubiquitination of FANCD2. Monoubiquitination of FANCD2 is required for resistance to the crosslinker mitomycin C. The ATR checkpoint kinase also regulates FANCD2 monoubiquitination and potentially links FANCD2 to the detection of DNA damage at the replication fork. The goal of this study is to understand the regulation and function of FANCD2 monoubiquitination, and thereby to better understand the collective role of FA proteins in promoting normal hematopoiesis. Certain FA proteins are mutated in spontaneous cancers in the general population, so these studies are also relevant to understanding how cancer develops. The specific aims of the study are as follows: 1) Delineate mechanisms of the ATR-dependent regulation of FANCD2 monoubiquitination. How, and whether, MCM7 and Rad17, along with Rad9, cooperate to couple FANCD2 monoubiquitination to the detection of DNA damage at the replication fork will be determined. ShRNA-mediated suppression of these proteins in primary human CD34+ cells, and expression of mutants in an immortalized myeloid precursor line, will be utilized. Whether these DNA damage sensors prevent chromosomal instability and apoptosis in response to MMC will also be determined. 2) Determine whether defects in FANCD2 monoubiquitination and the assembly of FANCD2 foci results in chromosomal instability and sensitivity to MMC in CD34+ hematopoietic stem/progenitor cells. FANCD2 monoubiquitination will be inhibited by shRNA-mediated suppression of FANCA. Also, mechanisms involved in the recruitment of FANCD2 to blocked replication forks will be elucidated using real-time microscopy in cells reconstituted with EGFP- FANCD2. PROJECT NARRATIVE It is increasingly clear that alterations in some Fanconi anemia pathway proteins are involved in some spontanous cancers seen in the general population. Thus the studies described in this application may shed new light on both normal and abnormal stem cell function and on cancer formation.

摘要 Fanconi贫血(FA)是一种导致进行性骨髓衰竭的多基因疾病 而且有很强的骨髓增生异常综合征和白血病倾向。FA蛋白是 通过一种基本未知的机制参与细胞对DNA损伤的反应。一颗核子 FANCD2的单素化需要至少8个FA蛋白的复合体。 FANCD2的单素化是产生对交联剂丝裂霉素C抗性所必需的 ATR检查点激酶也调节FANCD2的单泛素化和潜在的连接 FANCD2用于检测复制分叉处的DNA损伤。这项研究的目标是 了解FANCD2单素化的调节和功能，从而更好地 了解FA蛋白在促进正常造血中的集体作用。某些FA 在普通人群中，自发性癌症中的蛋白质会发生突变，因此这些研究是 也与了解癌症是如何发展有关的。这项研究的具体目的如下 以下是： 1)阐明ATR依赖的FANCD2单素化调控机制。 MCM7和Rad17以及Rad9如何以及是否合作偶联FANCD2 将确定在复制叉处检测DNA损伤的单素化。 ShRNA介导的这些蛋白在原代人CD34+细胞中的抑制及表达 永生化的髓系前体系中的突变体，将被利用。这些DNA是否 损伤传感器防止染色体不稳定和细胞凋亡对MMC的反应也将 要下定决心。 2)确定FANCD2单素化和FANCD2焦点组装中是否存在缺陷 CD34+造血细胞染色体不稳定性及对MMC敏感性的研究 干细胞/祖细胞。ShRNA介导的FANCD2单素化将受到抑制 对FANCA的抑制。此外，参与招募FANCD2的机制受阻 在用EGFP重组的细胞中，将使用实时显微镜来阐明复制分叉。 FANCD2.项目叙事 越来越清楚的是，一些Fanconi贫血途径蛋白的改变参与了 一些自发性癌症见于普通人群。因此，在本文件中描述的研究 应用可能为正常和异常干细胞功能以及癌症提供新的线索 队形。