The Role of And-1 in DNA Damage Response

描述（由申请人提供）：DNA损伤反应（DDR）是一种复杂的信号转导途径，其对于通过抵消DNA损伤的有害作用来维持基因组稳定性和预防致癌作用极其重要。在复制应激或紫外线照射下，DDR的主要事件是激活激酶ATR和Chk 1，进而触发多个生理过程，包括DNA合成抑制、DNA修复、细胞周期延迟、转录等。ATR在Chk 1激活和DNA合成抑制中起关键作用，以响应DNA损伤。然而，ATR如何与各种DDR组分协调以激活Chk 1以及ATR如何靶向复制装置以抑制DNA合成在哺乳动物细胞中仍然是未知的。我们已经证明，人类和-1作为复制体的关键组成部分，在复制叉的DNA复制。我们现在有可靠的初步数据表明，And-1也参与了对DNA损伤的检查点和DNA合成的调节。因此，阐明And-1在DDR中的作用将填补DDR信号传导的关键知识空白。在我们最近发表的工作和我们广泛的新数据的基础上，在这个提案中，我们描述了一系列创新的，假设驱动的研究，以阐明人类和-1如何调节检查点激活和DNA合成，以响应紫外线照射和复制应激。首先，我们将使用生化和结构分析来确定And-1如何被招募到DNA损伤位点的机制。其次，我们将确定And-1如何作为一个独特的复制体组件，通过影响多个关键DDR组件之间的相互作用，包括ATR，Rad 17，Claspin和Timeless-Tipin在停滞的复制叉使用分子和结构分析来调节检查点激活。 最后，我们将通过实施一种创新的方法来确定And-1在DNA损伤时控制DNA合成的机制，该方法结合了多种尖端技术，包括单分子分析，iPOND和质谱法。鉴于靶向DNA损伤反应的电路一直是抗癌药物开发的关键焦点，拟议的工作不仅将通过识别新组分和获得对DNA损伤反应的深入机制理解来推进该领域，而且还为我们提供了开发靶向And-1或And-1依赖过程的高度特异性抗癌疗法的新策略。

DESCRIPTION (provided by applicant): DNA damage response (DDR) is a complex signal transduction pathway, which is extremely important to maintain genomic stability and prevent carcinogenesis by counteracting the deleterious effects of DNA damage. In response to replication stress or UV irradiation, the primary event of DDR is to activate kinases ATR and Chk1, which in turn trigger multiple physiological processes, including DNA synthesis inhibition, DNA repair, and cell cycle delay, transcription, etc. ATR plays a key role in Chk1 activation and DNA synthesis inhibition in response to DNA damage. However, how ATR coordinates with various DDR components to activate Chk1 and how ATR targets replication apparatus to inhibit DNA synthesis remain largely unknown in mammalian cells. We have demonstrated that human And-1 acts as a key component of replisome at replication forks for DNA replication. We now have solid preliminary data indicating that And-1 is also involved in the regulation of checkpoint and DNA synthesis in response to DNA damage. Thus, elucidating the role of And-1 in DDR will fill in critical knowledge gaps of DDR signaling. Building upon our recently published work and our extensive new data, in this proposal we described a series of innovative, hypothesis-driven studies to elucidate how human And-1 regulates checkpoint activation and DNA synthesis in response to UV irradiation and replication stress. First, we will determine the mechanism of how And-1 is recruited to DNA damage sites using both biochemical and structural analyses. Second, we will determine how And-1 acts as a unique replisome component to regulate checkpoint activation by impacting interplay among multiple key DDR components including ATR, Rad17, Claspin, and Timeless-Tipin at stalled replication forks using molecular and structural analyses. Finally, we will determine mechanism by which And-1 governs DNA synthesis in response to DNA damage by carrying out an innovative approach that combines multiple cutting edge technologies including single-molecule analyses, iPOND, and mass spectrometry. Given that targeting the circuitry of DNA damage response has been a key focus for the development of anti-cancer drugs, proposed work will not only advance the field by identifying new components and acquiring in-depth mechanistic understanding of DNA damage response, but also provide us with new strategies for the development of highly specific anti-cancer therapies targeting And-1 or And-1-dependent processes.