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Fanconi贫血（FA）表型以基因组不稳定性和对DNA损伤的高敏性为标志。 FA蛋白参与将FA途径与其他更明确的途径并置的复合物，该途径参与细胞对DNA损伤的反应，例如ATR，BRCA1和RAD51。最近的工作已将FA生物学的重点和这些蛋白质的正常功能转移到同源重组途径上。然而，由于FA蛋白很少有已知的功能基序，因此在正常的FA生物化学或FA蛋白对造血和白血病的贡献的方式中尚待确定。对FA的生化途径的洞察力很重要，因为这将导致一种机制，即基因组不稳定性发生，这是癌症的基本原因。这种理解使预防癌症的干预措施以及对癌症治疗的基因组不稳定性的操纵。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性的机制，并了解FA蛋白的正常功能。提出的研究的中心假设是，fancd2充当信号转导蛋白与下游效应子（如MSH2，不匹配修复蛋白和MCM2-7复合物）相互作用，这对于复制机械的原始载荷至关重要。我们的工作表明，这两个过程都取决于上游磷酸化事件。为了测试我们的中心假设并执行此应用，我们将追求以下特定目的：目标＃1：确定fancd2-MSH2相互作用的功能后果，AIM＃1的工作假设是FANCD2-MSH2相互作用对于正常的DNA损伤响应很重要。我们的数据表明，MSH2敲低不仅会导致DNA损伤超敏反应，还会使FANCD2和MSH2的双重敲低矛盾地导致细胞抗性。 AIM＃2：确定FANCD2-MLH1相互作用的功能结果AIM 2的工作假设是FANCD2-MLH1相互作用对于正常的DNA损伤响应很重要。我们表明，MLH1损失不会影响FANCD2单数元素化，但确实可以阻止信号传播到染色质和随后对DNA交叉链接器的敏感性提高。 AIM＃3：证明FANCD2-MCM2-7相互作用的功能后果是AIM＃2的工作假设是，FANCD2-MCM2-7相互作用通过在S阶段响应DNA损害来调节复制复制复合络合物的组装。我们的数据表明，与MCM亚基的一个子集结合会导致抑制复制复合物组件。了解FA途径将进一步阐述关键的维修途径，并洞悉针对癌症治疗的途径。 公共卫生相关性：Fanconi贫血（FA）是一种癌敏感性的遗传疾病，其解剖导致研究了DNA损伤反应和同源重组修复。我们发现，FA途径中的中央蛋白质FANCD2通过MSH2和MLH1以及复制旋转酶MCM2-7与不匹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。对这些途径的研究将使我们不仅了解DNA修复和反应的基本机制，而且还将发现途径，从而可以调节癌症的机制和对癌症治疗的反应。