FANCD2 interaction with mismatch repair proteins and MCM2-7

描述（由申请人提供）：范可尼贫血（FA）在细胞水平上的表型以基因组不稳定和对DNA损伤的超敏性为特征。FA蛋白参与复合物，将FA通路与其他更明确的参与细胞对DNA损伤反应的通路并列，如ATR、BRCA1和RAD51。最近的工作已经将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，在正常的FA生物化学或FA蛋白促进造血和白血病的方式中，仍有许多有待确定，因为FA蛋白含有很少已知的功能基序。深入了解FA的生化途径是很重要的，因为这将导致基因组不稳定发生的机制，这是癌症的基本原因。这一认识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。我的实验室的长期目标是定义基因组不稳定的机制，使用FA作为模型，以及了解FA蛋白的正常功能。该研究的核心假设是FANCD2作为一种信号转导蛋白，与下游效应蛋白如MSH2、错配修复蛋白和MCM2-7复合物相互作用，这对复制机制的起源前装载至关重要。我们的工作表明，这两个过程都依赖于上游磷酸化事件。为了验证我们的中心假设并开展这项应用，我们将追求以下具体目标：目标#1：确定FANCD2-MSH2相互作用的功能后果。目标#1的工作假设是FANCD2-MSH2相互作用对正常的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的双重敲低也会导致细胞耐药。目标2：确定FANCD2-MLH1相互作用的功能后果目标2的工作假设是FANCD2-MLH1相互作用对正常的DNA损伤反应很重要。我们发现MLH1缺失不影响FANCD2单泛素化，但确实阻止了信号向染色质的传播，并随后增加了对DNA交联剂的敏感性。目标3：证明FANCD2-MCM2-7相互作用的功能后果目标2的工作假设是FANCD2-MCM2-7相互作用通过响应S期DNA损伤来调节起源处复制复合体的组装。我们的数据表明，与MCM亚基的一个亚基结合会抑制复制复合体的组装。了解FA通路将进一步阐明关键的修复通路，并为靶向癌症治疗的途径提供见解。

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.