Investigate the DNA damage response pathway of Fanconi anemia and BRCA proteins

研究范可尼贫血和 BRCA 蛋白的 DNA 损伤反应途径

• 批准号: 10467896
• 负责人: Weidong Wang
• 金额: $ 2.78万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467896
• 关键词: Aging; Atrophic condition of skin; BACH1 gene; BRCA1 gene; BRCA2 gene; Bilateral; Binding; CDC45L gene; Cancer-Predisposing Gene; Catalytic Domain; Cataract; Cell Nucleus; Cells; Characteristics; Chromatin; Collaborations; Complement; Complex; Congenital Abnormality; Core Protein; Cruciform DNA; Crystallization; DNA; DNA Binding; DNA Crosslinking; DNA Damage; DNA Interstrand Crosslinking; DNA Repair; DNA Structure; DNA biosynthesis; DNA replication fork; Data; Defect; Diabetes Mellitus; Disease; FANCB; FANCD2 protein; Fanconi Anemia Complementation Group A Protein; Fanconi Anemia pathway; Fanconi anemia protein; Fanconi' s Anemia; Frequencies; Gene Conversion; Genes; Genome Stability; Genomics; Goals; Hereditary Disease; Histone Fold; Human; In Situ; Lesion; Link; Malignant Neoplasms; Mediating; Mitomycin C; Modification; Monoubiquitination; Mutate; Names; Normal Cell; Nuclear; Orthologous Gene; Osteopenia; Osteoporosis; PALB2 gene; Pancytopenia; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Predisposition; Premature Ovarian Failure; Premature aging syndrome; Process; Proteins; Reaction; Reporting; Resistance; Role; Route; Signal Transduction; Spottings; Structure; Tissues; Transducers; Ubiquitin; Work; Yang; Yeasts; base; cancer risk; crosslink; genome integrity; helicase; homologous recombination; in vitro activity; in vivo; information model; malignant breast neoplasm; new therapeutic target; novel; novel therapeutic intervention; premature; protein function; recruit; reduced muscle mass; replication stress; response; ubiquitin ligase; xeroderma pigmentosum group A complementing protein

Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. The disease has also been considered by some as a segmental progeriod entity, as FA patients develop a range of tissue-specific premature onset and accelerated aging phenotypes, including bilateral cataracts, skin atrophy, reduced muscle mass, premature ovarian failure, higher frequency of osteoporosis and osteopenia, and diabetes. Sixteen genes have now been described that are mutated to cause FA. Three of them are discovered by our group. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in this pathway is modification of two FA proteins, FANCD2 and FANCI. The modification, monoubiquitylation, results in redistribution of FANCD2-FANCI to specific spots in the nucleus where BRCA proteins also localize. When we initiated this project in 2001, five FA proteins (FANCA, -C, -E, -F, and -G) were found to interact with each other to form a multiprotein nuclear complex, the FA core complex. This complex functions upstream in the pathway and is required for FANCD2-FANCI monoubiquitylation. We purified the FA core complex and found that it contains at least eight new components in addition to the five known FA proteins. We have characterized these new components and shown that they are important for the FA-associated DNA damage response pathway, as summarized below. One new component, termed FANCL, possesses ubiquitin ligase activity in vitro and is essential for FANCD2-FANCI monoubiquitylation in vivo. FANCL is defective in a group of Fanconi anemia patients, and therefore represents a novel Fanconi anemia gene. FANCL plays a crucial role in the Fanconi anemia pathway as the catalytic subunit for monoubiquitylation and FANCD2-FANCI. FANCL might be a potential target for new therapeutic intervention. The 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). FANCB is X-linked and present in only one active copy in normal cells. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability. The 250 Kda subunit of the FA core complex, named FANCM, is mutated in FA patients of a new complementation group, FA-M. FANCM has a conserved helicase domain and a DNA remodeling activity. FANCM has at least three important roles in the FA DNA damage response pathway. First, it plays a structural role, allowing assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM translocates and remodels various DNA structures, which are important for subsequent DNA repair. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. We identified the 100 Kda subunit of the FA core complex, FAAP100, and our collaboration with another group shows that it is also defective in an FA patient and required for FANCD2 monoubiquitination. We identified the 24 Kda subunit of the FA core complex, termed FAAP24, which forms a heterodimer with FANCM. FAAP24 can recognize structured DNA that mimics intermediates generated during DNA replication. Moreover, it can target FANCM to such structures. Cells depleted of FAAP24 show phenotypes that are characteristics of FA cells. We collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. In mechanistic studies, we demonstrated that FANCM possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimiics intermediates generated during homologous recombination or at stalled replication forks. We found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA crosslinking drug, mitomycin C (MMC), but not for the monoubiquitination of FANCD2-FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP- dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair. We identified two new components in the FA core complex, MHF1 and MHF2. These two proteins form a histone-fold heterodimer that associates with FANCM to form a DNA-remodeling complex conserved from yeast to human. MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. Notably, the yeast orthologs of these proteins also function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human. We showed that another FA protein, FANCJ, becomes hyperphosphorylated in response to DNA damage. We are investigating if this phosphorylation regulates activity of FANCJ in DNA repair. We collaborated with Dr. Lei Li's lab to develop a chromatin-IP-based strategy termed eChIP and elucidated how various FA proteins are recruited to the interstrand DNA crosslinks that block replication. We found that BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. We found that the FA network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a new component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network. We have worked with Lei Li's lab and showed that FANCM and FAAP24 can work either cooperatively or in parallel pathways to activate the FA network and protect genome stability. We have worked with Michael Seidman's group to show that DNA ICLs are not absolute blocks for replication, and one function of FANCM-MHF complex is to promote replication machinery to traverse the ICLs. Moreover, we found that the BLM complex is also required for this traverse activity. More recently, we and our collaborators found that FANCM can directly interact with replication machinery to promote the traverse reaction. We have worked with Wei Yang's lab and solved the crystal structure of MHF with or without its interaction domain of FANCM. We found that FANCM remodels the structure of MHF to recognize branched DNA, such as replication forks or Holliday junctions, and to protect genome stability.

范可尼贫血（FA）是一种隐性遗传性疾病，其特征是先天性缺陷、骨髓衰竭和癌症易感性。该疾病也被一些人认为是一种节段性早衰实体，因为 FA 患者会出现一系列组织特异性的过早发病和加速衰老表型，包括双侧白内障、皮肤萎缩、肌肉质量减少、卵巢早衰、骨质疏松症发生率较高以及骨质减少和糖尿病。目前已有 16 个基因被描述为导致 FA 的突变。其中三个是我们小组发现的。最近的证据表明，FA 蛋白在 DNA 损伤反应途径中发挥作用，涉及乳腺癌易感基因 BRCA1 和 BRCA2 产生的蛋白质。该途径的关键步骤是修饰两种 FA 蛋白 FANCD2 和 FANCI。这种修饰（单泛素化）导致 FANCD2-FANCI 重新分布到细胞核中 BRCA 蛋白也定位的特定位点。当我们于 2001 年启动该项目时，发现五种 FA 蛋白（FANCA、-C、-E、-F 和 -G）彼此相互作用，形成多蛋白核复合物，即 FA 核心复合物。该复合物在通路上游发挥作用，是 FANCD2-FANCI 单泛素化所必需的。我们纯化了 FA 核心复合物，发现除了 5 种已知的 FA 蛋白外，它还至少含有 8 种新成分。我们对这些新成分进行了表征，并表明它们对于 FA 相关的 DNA 损伤反应途径很重要，总结如下。 一种称为 FANCL 的新成分在体外具有泛素连接酶活性，并且对于体内 FANCD2-FANCI 单泛素化至关重要。 FANCL在一组范可尼贫血患者中存在缺陷，因此代表了一种新的范可尼贫血基因。 FANCL 作为单泛素化和 FANCD2-FANCI 的催化亚基，在范可尼贫血途径中发挥着至关重要的作用。 FANCL可能是新治疗干预的潜在目标。 Fanconi贫血核心复合体的95 kd亚基在FA互补B组患者中存在缺陷（该基因被命名为FANCB）。 FANCB 是 X 连锁的，在正常细胞中仅存在一个活性拷贝。因此，FANCB 可能是维持基因组稳定性的机制中的一个脆弱目标。 FA 核心复合物的 250 Kda 亚基（称为 FANCM）在新的互补组 FA-M 的 FA 患者中发生突变。 FANCM 具有保守的解旋酶结构域和 DNA 重塑活性。 FANCM 在 FA DNA 损伤反应途径中至少具有三个重要作用。首先，它发挥结构作用，允许 FA 核心复合物组装，因为如果没有它，几种 FA 蛋白的核定位和稳定性就会有缺陷。其次，FANCM 易位和重塑各种 DNA 结构，这对于随后的 DNA 修复很重要。第三，FANCM 因 DNA 损伤而过度磷酸化，这表明它可以作为信号转导器，通过该信号转导器来调节核心复合物的活性。 我们鉴定了 FA 核心复合物 FAAP100 的 100 Kda 亚基，并且我们与另一个小组的合作表明，FA 患者中它也有缺陷，并且是 FANCD2 单泛素化所必需的。 我们鉴定了 FA 核心复合物的 24 Kda 亚基，称为 FAAP24，它与 FANCM 形成异二聚体。 FAAP24 可以识别模仿 DNA 复制过程中产生的中间体的结构化 DNA。此外，它还可以将 FANCM 瞄准此类结构。 FAAP24 耗尽的细胞显示出 FA 细胞特征的表型。 我们与其他实验室合作证明，BRCA2 的伙伴 PALB2 是范可尼贫血补充 N 组患者的基因缺陷。 在机制研究中，我们证明 FANCM 在合成的四向连接 DNA 上具有不依赖于 ATP 的结合活性和依赖于 ATP 的双向分支点易位活性，该活性模仿同源重组期间或停滞复制叉时产生的中间体。我们发现 FANCM 的 ATP 依赖性活性是细胞对 DNA 交联药物丝裂霉素 C (MMC) 的耐药性所必需的，但 FANCD2-FANCI 的单泛素化不需要。相反，单泛素化需要 FANCM 的整个解旋酶结构域，该结构域具有 ATP 依赖性和独立活性。这些数据与 FANCM 及其相关 FA 核心复合物在 FA 途径中参与单泛素化信号传导和随后的 DNA 修复的情况一致。 我们在 FA 核心复合物中发现了两个新成分：MHF1 和 MHF2。这两种蛋白形成组蛋白折叠异二聚体，与 FANCM 结合形成从酵母到人类保守的 DNA 重塑复合物。 MHF 通过 FANCM 刺激 DNA 结合和复制叉重塑。在细胞中，FANCM 和 MHF 被迅速募集到因 DNA 链间交联而停滞的分叉上，两者都是细胞抵抗此类损伤所必需的。值得注意的是，这些蛋白质的酵母直系同源物还共同发挥作用，抵抗 MMS 诱导的 DNA 损伤，并促进受阻复制叉处的基因转换。因此，FANCM-MHF 是一种重要的 DNA 重塑复合物，可保护从酵母到人类的复制叉。 我们发现另一种 FA 蛋白 FANCJ 会因 DNA 损伤而过度磷酸化。我们正在研究这种磷酸化是否调节 FANCJ 在 DNA 修复中的活性。 我们与李磊博士的实验室合作开发了一种基于染色质 IP 的策略，称为 eChIP，并阐明了如何将各种 FA 蛋白招募到阻止复制的链间 DNA 交联中。我们发现 BRCA 相关 FA 蛋白（BRCA2、FANCJ/BACH1 和 FANCN/PALB2）需要复制才能实现交联关联，而 FA 核心和 I/D2 复合物则不需要。 FANCD2（而非 FANCJ 和 FANCN）需要 FA 核心复合体来进行招募。 FA 核心复合物需要核苷酸切除修复蛋白 XPA 和 XPC 才能结合。因此，FA 蛋白参与由 DNA 复制状态控制的不同 DNA 损伤反应机制。 我们发现 FA 网络受到泛素信号级联的调节，该级联由 RNF8 及其伙伴 UBC13 启动，并由 FA 核心复合物的新组件 FAAP20 介导。 FAAP20 优先结合 RNF8-UBC13 的泛素产物，这种泛素结合活性和 RNF8-UBC13 都是 FAAP20 招募到 ICL 所必需的。 RNF8 和 FAAP20 都是将 FA 核心复合物和 FANCD2 招募到 ICL 所必需的，而 RNF168 可以调节招募效率。 RNF8 和 FAAP20 是有效 FANCD2 单泛素化所必需的，这是 FA 网络的关键步骤； RNF8 和 FA 核心复合物以相同的途径发挥作用，促进细胞对 ICL 的抵抗。因此，RNF8-FAAP20 泛素级联对于将 FA 核心复合物招募到 ICL 以及 FA 网络的正常功能至关重要。 我们与李雷实验室合作，证明 FANCM 和 FAAP24 可以协同或并行工作，激活 FA 网络并保护基因组稳定性。 我们与 Michael Seidman 的团队合作，证明 DNA ICL 并不是复制的绝对阻碍，FANCM-MHF 复合物的功能之一是促进复制机制穿越 ICL。此外，我们发现 BLM 复合体也是这种遍历活动所必需的。最近，我们和我们的合作者发现 FANCM 可以直接与复制机制相互作用以促进遍历反应。 我们与杨伟实验室合作，解析了具有或不具有FANCM相互作用域的MHF的晶体结构。我们发现 FANCM 重塑了 MHF 的结构，以识别分支 DNA，例如复制叉或霍利迪连接，并保护基因组稳定性。