FANCJ dependent pathways in replication stress

项目概要： 在揭示复制应激中起作用的蛋白质和途径方面已经取得了很大的进展 反应特别是遗传性乳腺癌基因，以及范可尼贫血（FA）突变基因， 在复制应激反应中起作用。现在可以理解，它们在复制中的功能丧失 应激反应有助于相关肿瘤对化疗如顺铂的敏感性。 然而，BRCA-FA蛋白的独特功能在很大程度上是未知的。在这里，我们建议分析 DNA复制叉动力学，复制体成分，并确定具有特定缺陷的患者突变 在复制应激反应中。 为了确定细胞如何从有缺陷的复制转变为失调的复制， 表达不同突变形式的BRCA 1相关FANCJ，在乳腺癌/卵巢癌中也发生突变， 和FA。与BRCA 1类似，我们发现FANCJ的功能是保护复制分叉免于崩溃。 我们还发现，这种FANCJ分叉保护功能需要其与错配修复的直接交互 (MMR)蛋白质MLH 1。这一发现提供了关于为什么缺乏FANCJ-MLH 1相互作用的细胞不能 从复制压力中恢复。我们还鉴定了假定的功能获得性FANCJ突变体，如 BRCA 1-相互作用缺陷突变体，规避复制压力，保持叉完整，并赋予超 对复制应激诱导剂的抗性。在目标1中，我们将试图定义FANCJ互动如何指导 DNA复制叉动力学。鉴于FANCJ定位于复制叉，取代蛋白质， 解开DNA，我们假设破坏与失调的复制不仅反映了DNA的变化， 结构，而且在DNA复制叉中发现的蛋白质。在目标2中，我们将寻求确定如何 FANCJ有助于复制体的组成，在未受到挑战的和在强调复制叉。 复制应激诱导FANCJ蛋白相互作用和翻译后修饰的变化。一些 这些变化发生在我们发现的癌症患者的突变位点。在目标3中，我们将寻求 抗复制应激的FANCJ突变体诱导的变化揭示了调控FANCJ的机制 在癌症中失去的功能。总的来说，通过定义细胞如何屈服于-或生存-毒性DNA损伤， 通常会干扰复制，我们将深入了解细胞从 在癌症中有缺陷到失调的复制。 !

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. !