Mechanistic insights into ATR dependency during replication resulting from BRCA or ATM deficiency

DNA损伤反应（DDR）是影响生物学许多方面的基本过程。在哺乳动物细胞中，DDR影响细胞周期和DNA复制过程的调控、DNA修复和基因组稳定性、转录调控、衰老和细胞死亡，并与免疫应答相互作用。此外，各种DDR蛋白的缺陷可导致包括生长缺陷、加速衰老、癌症和神经退行性疾病在内的病理。为了应对发生的各种形式的DNA损伤，已经进化出各种DDR途径，这些途径共同包含数百种不同的蛋白质。众所周知，不同的DDR途径和成分在功能上是相互合作的，到目前为止，我们只看到了其中的一小部分，还有更多的有待发现。因此，更好地了解DDR信号和DDR通路之间的相互作用将为人类生物学、疾病和健康提供重要的新见解。DDR蛋白的一个关键作用是在DNA复制中，其中各种细胞压力可导致复制叉停滞，如果不有效处理，则通过复制叉“崩溃”产生细胞毒性DNA双链断裂（DSBs）。ATR（共济失调毛细血管扩张和Rad3相关）蛋白是s期发挥关键作用的关键DDR蛋白之一。ATR在复制应激反应中发挥着多种作用，包括稳定停滞的复制分叉，调节复制起始点的延迟启动，以及通过同源重组（homologous recombination， HR）修复dsb。最近，BRCA1和brca2的DSB修复独立机制也被确定，表明它们在停滞的复制分叉位点也发挥着不同的作用。我们计划深入了解BRCA1、BRCA2和ATM在复制应激反应中的作用机制，并了解在这三种DDR因子中缺乏任何功能的细胞中对ATR的依赖性。加强我们对BRCA1/2在停滞复制分叉中的作用的机制理解，不仅有助于我们对核心复制生物学的理解，而且还有助于深入了解HR蛋白如何能够影响其他DDR途径。重要的是，BRCA在复制分叉保护中的作用已经涉及到耐药性，这可以通过失去ATM来克服。新出现的数据还表明，ATR与BRCA1、BRCA2和ATM并不是上位性的，而是这些因素的缺乏导致对ATR抑制的敏感性显著增加。因此，了解这种相互作用背后的机制可以区分BRCA与ATR的联系与与ATM的相互作用，同时还可以深入了解BRCA在DSB修复和复制分叉稳定中的作用之间的差异和相似之处。Steve Jackson实验室成员最近的工作表明，基于CRISPR-Cas9的抗性筛选能够检测DDR中新的遗传和功能相互作用。因此，我们建议使用ATR抑制剂AZD6738的抗性筛选作为工具，使我们能够确定克服ATR依赖的机制，并通过这样做增强我们对BRCA1， BRCA2和ATM在复制应激反应途径中的作用的理解。在确定新的功能相互作用伙伴之后，我们将对这些相互作用背后的机制以及Jackson组过去和正在进行的筛选产生的机制进行详细的探索。除了建立我们对核心DDR和复制生物学的基本理解之外，进一步了解BRCA和ATM在其他DDR途径中的作用也可以帮助阿斯利康扩大DDR组合：1)为治疗干预提供新的合成致死伙伴；ii)预测对DDR治疗的耐药机制和iii)支持患者分层。

The DNA damage response (DDR) is a fundamental process that impacts on many aspects of biology. In mammalian cells, the DDR affects the regulation of the cell cycle and DNA replication processes, DNA repair and genomic stability, transcriptional regulation, senescence and cell death, and interplays with immune responses. Furthermore, defects in various DDR proteins can result in pathologies including growth defects, accelerated aging, cancer and neurodegenerative disease.To cope with the diverse forms of DNA lesions that occur, various DDR pathways have evolved that collectively comprise several hundred distinct proteins. It is known that that different DDR pathways and components functionally cooperate and we have so far only glimpsed a small proportion of these, with many more remaining to be discovered. Consequently, a better understanding of DDR signalling and the interplay between DDR pathways will provide important new insights into human biology, disease and health.One key role of DDR proteins is in DNA replication, where various cellular stresses can result in replication fork stalling and, if not effectively dealt with, generate cytotoxic DNA double strand breaks (DSBs) through replication fork 'collapse'. One of the key DDR proteins that plays pivotal functions during S-phase is the ATR (Ataxia Telangiectasia and Rad3 related) protein. ATR plays multiple roles in the replication stress response including the stabilization of stalled replication forks, regulation of late replication origin firing, and repair of DSBs by homologous recombination (HR). Recently, DSB repair-independent mechanisms for BRCA1 and 2 have also been identified, showing that they too play distinct roles at the site of stalled replication forks.We plan to gain mechanistic insight into the roles of BRCA1, BRCA2 and ATM in the replication stress response as well as understanding the dependency on ATR in cells that lack functionality in any of these three DDR factors. Enhancing our mechanistic understanding of BRCA1/2's role at stalled replication forks would not only contribute to our understanding of core replication biology, but would also provide insight into how HR proteins are able to influence other DDR pathways. Importantly, BRCA's role in replication fork protection has already been implicated in drug resistance, which can be overcome by a loss of ATM. Emerging data also suggest that ATR is not epistatic with BRCA1, BRCA2 and ATM, but rather deficiencies in these factors confer a significant increase in sensitivity to ATR inhibition. Understanding the mechanism(s) behind this interplay could therefore differentiate BRCA's link with ATR from its interaction with ATM, alongside providing insights into the differences and parallels between BRCA's role in DSB repair and in replication fork stabilisation.Recent work by members of the Steve Jackson lab has shown that CRISPR-Cas9 based resistance screens are able to detect novel genetic and functional interactions in the DDR. We therefore propose using resistance screens with the ATR inhibitor AZD6738 as a tool to allow us to identify mechanisms of overcoming ATR dependency, and in doing so enhance our understanding of the roles of BRCA1, BRCA2 and ATM in the replication stress response pathway. Following the identification of new functional-interaction partners, we will then undertake detailed probing of the mechanisms behind these interactions as well as those that have arisen from past and ongoing screens in the Jackson group.In addition to building upon our fundamental understanding of core DDR and replication biology, further understanding the role of BRCA and ATM in other DDR pathways could also help AstraZeneca's expanding DDR portfolio by i) suggesting novel synthetically lethal partners for therapeutic intervention; ii) predicting resistance mechanisms to DDR therapeutics and iii) supporting patient stratification.