FANCJ dependent pathways in replication stress

项目总结： 在揭示在复制应激中起作用的蛋白质和途径方面已经取得了很大进展 回应。尤其是遗传性乳腺癌基因，以及范可尼贫血(FA)中的基因突变。 在复制应激反应中起作用。现在可以理解，它们在复制中功能丧失 应激反应导致相关肿瘤对化疗药物的敏感性，如顺铂。 然而，BRCA-FA蛋白的不同功能在很大程度上是未知的。在这里，我们建议分析 DNA复制分叉动态、复制体组件，并识别具有特定缺陷的患者突变 在复制应激反应中。 为了定义细胞如何从缺陷复制转变为失调复制，我们设计了细胞 表达不同突变版本的BRCA1相关FANCJ在乳腺癌/卵巢癌中也发生突变 和FA.与BRCA1类似，我们已经发现FANCJ具有保护复制分叉免受崩溃的功能。 我们还发现，这种FANCJ叉形保护功能需要它与错配修复直接相互作用 (MMR)蛋白，MLH1。这一发现为缺乏FANCJ-MLH1相互作用的细胞未能 从复制压力中恢复。我们还鉴定了假定的功能增益FANCJ突变体，如 BRCA1-相互作用缺陷突变体，绕过复制压力，保持叉子完好无损，并提供超 对复制应激诱导剂的抗性。在目标1中，我们将寻求定义FANCJ互动如何指导 DNA复制分叉动力学。鉴于FANCJ定位于复制叉子，取代蛋白质，以及 解开DNA，我们假设干扰与失调的复制不仅反映了DNA的变化 结构，还有DNA复制叉处发现的蛋白质。在目标2中，我们将寻求确定如何 FANCJ对复制体的组成有贡献，无论是在未受到挑战的还是在应激的复制叉处。 复制应激诱导FANCJ蛋白相互作用和翻译后修饰的变化。其中一些 这些变化发生在我们发现的癌症患者突变的位置。在目标3中，我们将寻求产生 FANCJ耐复制胁迫突变体诱导FANCJ调控机制的改变 在癌症中丧失的功能。总而言之，通过定义细胞如何屈服于或存活于有毒的DNA损伤 这通常会干扰复制，我们将深入了解细胞从 在癌症中存在复制失调的缺陷。 好了！

Project Summary: Great progress has been made in uncovering the proteins and pathways that function in the replication stress response. In particular, the hereditary breast cancer genes, as well as genes mutated in Fanconi anemia (FA) function in the replication stress response. It is now understood that loss of their function in the replication stress response contributes to the sensitivity of associated tumors to chemotherapies, such as cisplatin. However, the distinct functions for the BRCA-FA proteins are largely unknown. Here, we propose to analyze DNA replication fork dynamics, replisome components, and identify patient mutations that have specific defects in the replication stress response. To define how a cell transitions from defective to dysregulated replication, we have engineered cells expressing different mutant versions of the BRCA1-associated FANCJ also mutated in breast/ovarian cancer and FA. Similar to BRCA1, we have uncovered that FANCJ functions to protect replication forks from collapse. We also found that this FANCJ fork protection function requires its direct interaction with the mismatch repair (MMR) protein, MLH1. This finding provides insight as to why cells lacking the FANCJ-MLH1 interaction fail to recover from replication stress. We have also identified putative gain-of-function FANCJ mutants, such as the BRCA1-interaction defective mutant, that circumvent replication stress, keep forks intact, and confer hyper- resistance to replication stress inducing agents. In Aim 1, we will seek to define how FANCJ interactions direct DNA replication fork dynamics. Given that FANCJ localizes to replication forks, displaces proteins, and unwinds DNA, we hypothesize that disrupted vs dysregulated replication will reflect not only changes in DNA structures, but also the proteins found at DNA replication forks. In Aim 2, we will seek to determine how FANCJ contributes to the composition of the replisome in both unchallenged and at stressed replication forks. Replication stress induces changes to FANCJ protein interactions and post-translation modifications. Some of these changes occur at sites we found to be mutated in cancer patients. In Aim 3, we will seek to generate FANCJ mutants resistant to replication stress induced changes to uncover mechanisms regulating FANCJ function that are lost in cancer. Collectively, by defining how cells succumb to- or survive- toxic DNA damage that normally interferes with replication, we will gain insight towards mechanisms transitioning cells from defective to dysregulated replication in cancer. !