FANCD2 interaction with mismatch repair proteins and MCM2-7

FANCD2 与错配修复蛋白和 MCM2-7 的相互作用

• 批准号: 8231274
• 负责人: Gary M. Kupfer
• 金额: $ 49.65万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-10 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231274
• 关键词: Affect; BRCA1 gene; Binding; Biochemical; Biochemical Pathway; Biochemistry; Biology; Cancer Etiology; Cancer Prevention Intervention; Cells; Chromatin; Complex; DNA; DNA Crosslinking Agent; DNA Damage; DNA Repair; Data; Defect; Dissection; Event; FANCD2 protein; Fanconi anemia protein; Fanconi' s Anemia; Genetic Recombination; Genomic Instability; Goals; Hematological Disease; Hematopoiesis; Hematopoietic Neoplasms; Hereditary Disease; Hypersensitivity; Laboratories; Lead; MCM2 gene; MLH1 gene; MSH2 gene; Malignant Neoplasms; Maps; Mismatch Repair; Modeling; Normal Cell; Pathway interactions; Pediatric Hematology/Oncology; Phenotype; Phosphorylation; Phosphorylation Site; Predisposition; Process; Proteins; Research; Resistance; S Phase; Signal Transduction; Testing; Work; cancer therapy; helicase; homologous recombination; human MCM2 protein; insight; leukemia; mutant; prevent; protein function; public health relevance; repaired; response

DESCRIPTION (provided by applicant): The Fanconi anemia (FA) phenotype on a cellular level is marked by genomic instability and hypersensitivity to DNA damage. FA proteins participate in complexes that juxtapose the FA pathway with other more defined pathways involved in cellular response to DNA damage, such as ATR, BRCA1, and RAD51. Recent work has served to shift focus of FA biology and the normal functions of these proteins onto homologous recombination pathways. However, much remains to be ascertained in normal FA biochemistry or about the manner in which FA proteins contribute to hematopoiesis and leukemia, as the FA proteins contain few known functional motifs. Insight into the biochemical pathway of FA is important because this would lead to a mechanism whereby genomic instability occurs, which is a basic cause of cancer. This understanding makes possible interventions for cancer prevention as well as manipulation of genomic instability for cancer therapy. The long-term goal of my laboratory is to define mechanisms of genomic instability, using FA as a model, as well as to understand the normal function of FA proteins. The central hypothesis for the proposed research is that FANCD2 acts as a signal transduction protein to interact with downstream effectors such as the MSH2, the mismatch repair protein, and the MCM2-7 complex, which is critical for pre-origin loading of the replication machinery. Our work suggests that both these processes depend on upstream phosphorylation events. To test our central hypothesis and carry out this application, we will pursue the following specific aims: Aim #1: Determine the functional consequence of FANCD2-MSH2 interaction The working hypothesis for Aim #1 is that FANCD2-MSH2 interaction is important for the normal DNA damage response. Our data have shown that not only does MSH2 knockdown result in DNA damage hypersensitivity but also double knockdown of both FANCD2 and MSH2 contradictorily results in cellular resistance. Aim #2: Determine the functional consequence of FANCD2-MLH1 interaction The working hypothesis for Aim #2 is that FANCD2-MLH1 interaction is important for the normal DNA damage response. We show that MLH1 loss does not affect FANCD2 monoubiquitylation but does prevent the propagation of signal to chromatin and subsequent increased sensitivity to DNA crosslinkers. Aim #3: Demonstrate the functional consequence of FANCD2-MCM2-7 interaction The working hypothesis for Aim #2 is that FANCD2-MCM2-7 interaction regulates the assembly of the replication complex at the origin by responding to DNA damage during S phase. Our data indicate that binding to a subset of the MCM subunits results in inhibition of replication complex assembly. Understanding the FA pathway will further elaborate a key repair pathway as well as lend insight into avenues of targeting cancer therapy. PUBLIC HEALTH RELEVANCE: Fanconi anemia (FA) is a genetic disease of cancer susceptibility whose dissection has led to the study of the DNA damage response and homologous recombinatorial repair. We have found that the central protein FANCD2 in the FA pathway interacts with the mismatch repair pathway via MSH2 and MLH1 as well the replicative helicase MCM2-7. We hypothesize that FANCD2 regulates the action of repair and replication through these proteins. Study of these pathways will allow us to not only understand basic mechanisms of DNA repair and response but also will uncover avenues whereby mechanisms of cancer and response to cancer therapy may be modulated.

描述（由申请人提供）：细胞水平上的范可尼贫血（FA）表型的特点是基因组不稳定性和对 DNA 损伤的超敏反应。 FA 蛋白参与复合物，将 FA 途径与参与细胞对 DNA 损伤反应的其他更明确的途径（例如 ATR、BRCA1 和 RAD51）并置。最近的工作已将 FA 生物学和这些蛋白质的正常功能的焦点转移到同源重组途径上。然而，由于 FA 蛋白含有很少的已知功能基序，因此在正常 FA 生物化学或 FA 蛋白促进造血和白血病的方式方面仍有许多待确定。深入了解 FA 的生化途径非常重要，因为这将导致基因组不稳定发生的机制，而这是癌症的基本原因。这种理解使得癌症预防的干预措施以及癌症治疗中基因组不稳定性的操纵成为可能。我实验室的长期目标是使用 FA 作为模型来定义基因组不稳定的机制，并了解 FA 蛋白的正常功能。拟议研究的中心假设是 FANCD2 作为信号转导蛋白与下游效应子（例如 MSH2、错配修复蛋白和 MCM2-7 复合物）相互作用，这对于复制机制的预起始加载至关重要。我们的工作表明这两个过程都依赖于上游磷酸化事件。为了测试我们的中心假设并实施该应用，我们将追求以下具体目标： 目标#1：确定 FANCD2-MSH2 相互作用的功能结果 目标#1 的工作假设是 FANCD2-MSH2 相互作用对于正常 DNA 损伤反应很重要。我们的数据表明，MSH2 敲低不仅会导致 DNA 损伤超敏反应，而且 FANCD2 和 MSH2 的双重敲低也会矛盾地导致细胞耐药。目标#2：确定 FANCD2-MLH1 相互作用的功能结果 目标#2 的工作假设是 FANCD2-MLH1 相互作用对于正常的 DNA 损伤反应很重要。我们发现 MLH1 丢失不会影响 FANCD2 单泛素化，但确实会阻止信号传播到染色质，并随后增加对 DNA 交联剂的敏感性。目标#3：展示 FANCD2-MCM2-7 相互作用的功能结果 目标#2 的工作假设是 FANCD2-MCM2-7 相互作用通过响应 S 期的 DNA 损伤来调节复制复合物在起点的组装。我们的数据表明，与 MCM 亚基的子集结合会导致复制复合物组装的抑制。了解 FA 途径将进一步阐述关键的修复途径，并有助于深入了解靶向癌症治疗的途径。 公共健康相关性：范可尼贫血 (FA) 是一种癌症易感性遗传病，对其的剖析引发了对 DNA 损伤反应和同源重组修复的研究。我们发现FA途径中的中心蛋白FANCD2通过MSH2和MLH1以及复制解旋酶MCM2-7与错配修复途径相互作用。我们假设 FANCD2 通过这些蛋白质调节修复和复制的作用。对这些途径的研究不仅使我们能够了解 DNA 修复和反应的基本机制，而且还将揭示可调节癌症机制和癌症治疗反应的途径。