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.

Fanconi贫血（FA）是一种隐性遗传疾病，其特征是先天性缺陷，骨髓衰竭和癌症敏感性。由于FA患者发展了一系列组织特异性的早产和加速衰老表型，包括双侧性白内障，皮肤萎缩，较低的卵巢肿块，卵巢早期衰竭，较高的骨质疏松症和骨质疏松症和糖尿病。 现在已经描述了15个基因，这些基因被突变为引起FA，这三个是由我们小组发现的。最近的证据表明，FA蛋白在DNA损伤反应途径中起作用，该途径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改两个FA蛋白FANCD2和FANCI。修饰，单位素化，导致fancd2-fanci重新分布到核中BRCA蛋白也定位的细胞核中的特定斑点。当我们在2001年启动该项目时，发现五种FA蛋白（FANCA，-C，-e，-f和-g）相互相互作用，形成了多蛋白核复合物，即FA核心复合物。该复合物在途径中的上游功能，是Fancd2-Fanci MonubiquityLation所必需的。我们纯化了FA核心复合物，发现除了五种已知的FA蛋白外，它至少包含七个新组件。我们已经表征了这些新组件，并表明它们对于与FA相关的DNA损伤响应途径很重要，如下所示。 FA核心复合物的一个新组成部分称为PHF9，在体外具有泛素连接酶活性，对于体内的fancd2-fanci monubiquitylation至关重要。 PHF9在一组Fanconi贫血患者中有缺陷，因此代表了一种新型的Fanconi贫血基因（FANCL）。我们的数据表明，PHF9在Fanconi贫血途径中起着至关重要的作用。 PHF9/FANCL的发现可能为新的治疗方式提供了潜在的目标。 然后，我们证明了Fanconi贫血核心复合物的95 kD亚基在FA补充B组患者中有缺陷（该基因称为FANCB）。 Fancb是X连锁的，仅在一个活动副本中显示。因此，粉丝可以代表维持基因组稳定性的机械中的脆弱靶标，因为与使其他常染色体FA基因失活所需的两个突变相比，它只需要一个突变就可以使粉丝失活。 我们证明了FA核心复合物的250 kDa亚基在新的补充组FA-M的FA患者中被突变。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损伤响应途径中至少具有三个重要作用。首先，它起着结构性的作用，允许将FA核心复合体组装，因为在不存在的情况下，几种FA蛋白的核定位和稳定性是有缺陷的。其次，FANCM易位并重塑了各种DNA结构，这对于随后的DNA修复很重要。第三，FANCM响应DNA损伤而被过度磷酸化，这表明它可以用作信号传感器，通过该信号传感器调节核心复合物的活性。 我们已经确定了FA核心复合物FAAP100的100 kDa亚基，并表明该蛋白是稳定性和fancd2-fanci复合物泛素化的关键功能所必需的。 我们已经确定了FA核心复合物的24 kDa亚基，称为FAAP24，该子络合物与FANCM形成异二聚体。 FAAP24可以识别模仿在DNA复制过程中产生的中间体的结构化DNA。此外，它可以将粉丝瞄准此类结构。耗尽FAAP24的细胞显示出是FA细胞特征的表型。我们的结果表明，FAAP24是FA核心复合物的组成部分， 我们还与其他实验室合作，证明了BRCA2的合作伙伴PALB2是Fanconi贫血补充组N患者的基因缺陷。 在进一步的机械研究中，我们证明了粉丝具有与ATP独立的结合活性和ATP依赖性的双向分支点易位活性在合成的四向连接DNA上，模仿了同源重组或停滞的复制叉时产生的中间体。我们发现，FANCM的ATP依赖性活性是对DNA交联药物丝裂霉素C（MMC）的耐药性所必需的，但对于FANCD2-FANCI的单次素化而不是。相比之下，单泛素化需要整个FANCM的解旋酶结构域，该结构域具有ATP依赖性和独立活动。这些数据与FANCM及其相关的FA核心复合物的参与在FA通路中的参与，这两种信号通过单样泛素化和随后的DNA修复。 我们确定了FA核心复合物MHF1和MHF2中的两个新组件。这两种蛋白质形成一个组蛋白折叠二聚体，该异二聚体与FANCM相关联，形成了从酵母到人的DNA复合复合物。 MHF刺激FANCM刺激DNA结合和复制叉重塑。在细胞中，FANCM和MHF迅速募集到DNA链间交叉链接停滞的叉子上，并且两者都是细胞对此类病变的耐药性所必需的。在脊椎动物中，FANCM-MHF与Fanconi贫血（FA）核心复合物相关联，促进了对DNA损伤的fancd2单次素数，并抑制了姐妹 - 染色剂交换。这些蛋白质的酵母直系同源物共同作用，以抵抗MMS诱导的DNA损伤并促进封闭的复制叉处的基因转化。因此，FANCM-MHF是一种必不可少的DNA造型复合物，可保护从酵母到人类的复制叉。 我们表明，另一种FA蛋白FANCJ会因DNA损伤而被过度磷酸化。我们正在研究这种磷酸化是否调节了FANCJ在DNA修复中的活性。 我们与Lei Li博士的实验室合作制定了一种基于染色质IP的策略，该策略称为ECHIP，并阐明了如何将各种FA蛋白招募到阻断复制的链链DNA交叉链接。我们发现与BRCA相关的FA蛋白（BRCA2，FANCJ/BACH1和FANCN/PALB2），但不需要FA核和I/D2复合物，需要复制其交联关联。 FANCD2，但不是Fancj和Fancn，需要FA Core Complex招募。 FA核心复合物需要核苷酸切除修复蛋白XPA和XPC的关联。因此，FA蛋白参与受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核心复合物的新组成部分。 FAAP20包含一个泛素结合基序，FA核心复合物需要定位于DNA损伤位点。 FAAP20的耗尽或灭活会减少粉状粉的单位化，这表明FAAP20是FA途径的重要参与者。 FANCM和MHF1-MHF2构成保守的DNA重塑机的事实促使我们对该复合物进行了晶体结构研究。到目前为止，我们还解决了MHF1-MHF2复合物的晶体结构与结合MHF的FANCM区域。该结构揭示了这三种蛋白质如何相互作用以形成复合物并识别DNA。 我们将继续识别和表征FA复合体的其他组件。最终的目标是分析DNA交联修复的完整途径，评估其在正常衰老中的作用以及在DNA修复缺陷疾病中。