FANCD2 interaction with mismatch repair proteins and MCM2-7

描述（由申请人提供）：细胞水平上的范可尼贫血（FA）表型以基因组不稳定性和对DNA损伤的超敏反应为特征。FA蛋白参与复合物，该复合物将FA途径与参与对DNA损伤的细胞反应的其他更明确的途径（例如ATR、BRCA 1和RAD 51）并列。最近的工作已经将FA生物学的焦点和这些蛋白质的正常功能转移到同源重组途径上。然而，在正常的FA生物化学或FA蛋白有助于造血和白血病的方式仍有待确定，因为FA蛋白含有很少的已知功能基序。深入了解FA的生化途径是重要的，因为这将导致基因组不稳定性发生的机制，这是癌症的基本原因。这种理解使得癌症预防的干预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以FA为模型，确定基因组不稳定性的机制，并了解FA蛋白的正常功能。这项研究的中心假设是，FANCD 2作为一种信号转导蛋白与下游效应物相互作用，如MSH 2、错配修复蛋白和MCM 2 -7复合物，这对复制机制的起始前加载至关重要。我们的工作表明，这两个过程依赖于上游磷酸化事件。为了检验我们的中心假设并实现这一应用，我们将追求以下具体目标：目标#1：确定FANCD 2-MSH 2相互作用的功能后果目标#1的工作假设是FANCD 2-MSH 2相互作用对正常DNA损伤反应是重要的。我们的数据表明，不仅MSH 2敲低导致DNA损伤超敏反应，而且FANCD 2和MSH 2的双重敲低矛盾地导致细胞抗性。目标二：目的#2的工作假设是FANCD 2-MLH 1相互作用对于正常的DNA损伤应答是重要的。我们表明，MLH 1的损失不影响FANCD 2单泛素化，但确实阻止了信号传播到染色质和随后的DNA交联剂的敏感性增加。目标3：目的#2的工作假设是FANCD 2-MCM 2 - 7相互作用通过响应S期期间的DNA损伤来调节复制复合物在起点的组装。我们的数据表明，结合到一个子集的MCM亚基的结果在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，并深入了解靶向癌症治疗的途径。 公共卫生相关性：范可尼贫血（FA）是一种癌症易感性遗传性疾病，其解剖导致了DNA损伤反应和同源重组修复的研究。我们发现FA通路中的中心蛋白FANCD 2通过MSH 2和MLH 1以及复制解旋酶MCM 2 -7与错配修复通路相互作用。我们推测FANCD 2通过这些蛋白调节修复和复制的作用。对这些通路的研究不仅可以使我们了解DNA修复和反应的基本机制，还可以揭示调节癌症机制和癌症治疗反应的途径。

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.