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: 8335892
• 负责人: Weidong Wang
• 金额: $ 39.19万
• 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/8335892
• 关键词: Aging; Aging-Related Process; Atrophic condition of skin; BRCA1 gene; BRCA2 gene; Bilateral; Binding; Cancer-Predisposing Gene; Catalytic Domain; Cataract; Cell Nucleus; Cells; Characteristics; Chromatin; Complex; Congenital Abnormality; DNA; DNA Binding; DNA Crosslinking; DNA Damage; DNA Interstrand Crosslinking; DNA Repair; DNA Structure; DNA biosynthesis; DNA crosslink; DNA replication fork; Data; Defect; Diabetes Mellitus; Disease; Fanconi anemia protein; Fanconi' s Anemia; Frequencies; Gene Conversion; Genes; Genome; Genome Stability; Goals; Histone Fold; Human; Inherited; Lesion; Link; Malignant Neoplasms; Mitomycins; Modality; Modeling; Modification; Monoubiquitination; Mutate; Mutation; Names; Nuclear; Orthologous Gene; Osteopenia; Osteoporosis; Pancytopenia; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Predisposition; Premature Ovarian Failure; Premature aging syndrome; Process; Proteins; Recruitment Activity; Resistance; Role; Route; Signal Transduction; Sister Chromatid Exchange; Site; Spottings; Structure; Tissues; Transducers; Ubiquitin; Vertebrates; Yeasts; base; cancer risk; crosslink; helicase; homologous recombination; in vitro activity; in vivo; male; malignant breast neoplasm; muscle form; new therapeutic target; normal aging; novel; premature; protein function; protein structure function; repaired; 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. Fifteen genes have now been described that are mutated to cause FA, three 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 that 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 seven 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 of the FA core complex, termed PHF9, possesses ubiquitin ligase activity in vitro and is essential for FANCD2-FANCI monoubiquitylation in vivo. PHF9 is defective in a group of Fanconi anemia patients, and therefore represents a novel Fanconi anemia gene (FANCL). Our data suggest that PHF9 plays a crucial role in the Fanconi anemia pathway as the catalytic subunit for monoubiquitylation and FANCD2-FANCI. The discovery of PHF9/FANCL might provide a potential target for new therapeutic modalities. We then showed that 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. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability, because it will take only one mutation to inactivate FANCB in males, compared to two mutations required to inactivate other autosomal FA genes. We demonstrated that the 250 Kda subunit of the FA core complex is mutated in FA patients of a new complementation group, FA-M. The gene was named FANCM. 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 have identified the 100 Kda subunit of the FA core complex, FAAP100, and shown that this protein is required for stability and a key function of the complexmonoubiquitination of FANCD2-FANCI. We have 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. Our results demonstrate that FAAP24 is an integral component of the FA core complex, We also collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. In further 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 mimics 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. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins 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 have recently identified FAAP20 as a new component of the FA core complex. FAAP20 contains an ubiquitin binding motif and is required for FA core complex to localize to DNA damage sites. Depletion or inactivation of FAAP20 reduces monoubiquitylation of FANCD2-FANCI, indicating that FAAP20 is an important player of the FA pathway. The fact that FANCM and MHF1-MHF2 constitute a conserved DNA remodeling machine prompted us to perform crystal structure studies of this complex. So far, we have also solved the crystal structure of MHF1-MHF2 complex in conjunction with the region of FANCM that binds MHF. The structure reveals how these three proteins interact with each other to form a complex and recognizes DNA. We are continuing to identify and characterize additional components of the FA complex. The eventual goal is to analyze the full pathway of DNA crosslink repair, assess its role during normal aging, and in DNA repair-deficient diseases.

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 15 种基因被描述为导致 FA 的突变，其中 3 种是我们小组发现的。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 7 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。 FA 核心复合物的一种新成分称为 PHF9，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。 PHF9 在一组范可尼贫血患者中存在缺陷，因此代表一种新的范可尼贫血基因（FANCL）。我们的数据表明，PHF9 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。 PHF9/FANCL 的发现可能为新的治疗方式提供潜在的靶点。 然后我们发现，FA 互补 B 组患者中 Fanconi 贫血核心复合体的 95 kd 亚基存在缺陷（该基因被命名为 FANCB）。 FANCB 是 X 连锁的，并且仅存在于一份活动副本中。因此，FANCB 可能是维持基因组稳定性机制中的一个脆弱靶标，因为与使其他常染色体 FA 基因失活所需的两个突变相比，仅需一个突变即可使男性 FANCB 失活。 我们证明了 FA 核心复合物的 250 Kda 亚基在新的互补组 FA-M 的 FA 患者中发生突变。该基因被命名为 FANCM。 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。 我们已经鉴定了 FA 核心复合物 FAAP100 的 100 Kda 亚基，并表明该蛋白是 FANCD2-FANCI 复合物单泛素化的稳定性和关键功能所必需的。 我们已经鉴定了 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。我们的结果表明 FAAP24 是 FA 核心复合物的一个组成部分， 我们还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。 在进一步的机制研究中，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，模拟同源重组过程中或复制叉停滞时产生的中间体。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。在脊椎动物中，FANCM-MHF 与范可尼贫血 (FA) 核心复合物结合，促进 FANCD2 单泛素化以应对 DNA 损伤，并抑制姐妹染色单体交换。这些蛋白质的酵母直系同源物共同发挥作用，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。 我们最近将 FAAP20 确定为 FA 核心复合体的新组成部分。 FAAP20 含有泛素结合基序，是 FA 核心复合物定位到 DNA 损伤位点所必需的。 FAAP20 的耗尽或失活会降低 FANCD2-FANCI 的单泛素化，表明 FAAP20 是 FA 途径的重要参与者。 FANCM 和 MHF1-MHF2 构成保守的 DNA 重塑机器这一事实促使我们对该复合物进行晶体结构研究。到目前为止，我们还结合FANCM结合MHF的区域解决了MHF1-MHF2复合物的晶体结构。该结构揭示了这三种蛋白质如何相互作用形成复合物并识别 DNA。 我们正在继续识别和表征 FA 复合体的其他组成部分。最终目标是分析 DNA 交联修复的完整途径，评估其在正常衰老和 DNA 修复缺陷疾病中的作用。