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复制过程的调节、DNA修复和基因组稳定性、转录调节、衰老和细胞死亡，并与免疫反应相互作用。此外，各种DDR蛋白的缺陷可能导致包括生长缺陷、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，这些途径总共由数百种不同的蛋白质组成。众所周知，不同DDR途径和组件在功能上是合作的，到目前为止，我们只瞥见了其中的一小部分，还有更多的东西有待发现。因此，更好地了解DDR信号以及DDR信号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，各种细胞应激可导致复制叉停滞，如果不能有效处理，则通过复制叉“崩溃”产生细胞毒性DNA双链断裂(DSB)。在S期起关键作用的DDR蛋白之一是ATR(共济失调、毛细血管扩张和RAD3相关)蛋白。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叉子，调节晚期复制起点的启动，以及通过同源重组(HR)修复DSB。最近，BRCA1和BRCA2的DSB修复独立机制也被发现，表明它们在停滞的复制分叉位置也发挥着不同的作用。我们计划从机械性的角度深入了解BRCA1、BRCA2和ATM在复制应激反应中的作用，以及理解在这三个DDR因子中缺乏功能的细胞对ATR的依赖。加强我们对BRCA1/2‘S在停滞复制分叉中的作用的机制的理解，不仅有助于我们理解核心复制生物学，也将有助于我们洞察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与耐药性有关，这种耐药性可以通过ATM的丢失来克服。新的数据还表明，ATR与BRCA1、BRCA2和ATM不是上位性的，而是这些因素的缺陷使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(S)可以区分BRCA与ATR的联系和与ATM的相互作用，并提供对BRCA在DSB修复和复制叉稳定中作用之间的差异和相似之处的见解。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，我们建议使用带有ATR抑制剂AZD6738的抗性筛选作为一种工具，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。在确定了新的功能相互作用合作伙伴后，我们将对这些相互作用背后的机制以及杰克逊集团过去和正在进行的筛查中出现的机制进行详细的探索。除了建立在我们对核心DDR和复制生物学的基本了解之外，进一步了解BRCA和ATM在其他DDR途径中的作用也有助于阿斯利康扩大DDR产品组合，方法包括：i)为治疗干预建议新的合成致命合作伙伴；ii)预测DDR疗法的耐药性机制；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.