PAR binding to Chk1 regulates development and tissue homeostasis

单链DNA断裂(SSB)和复制应激激活PARP1，PARP1催化聚(ADP-核糖)(PAR)聚合物的形成以及靶蛋白的PAR化。PAR被假设作为信号传递给其他细胞靶点，包括修复蛋白。PAR化和PARP1已被证明在多种细胞过程中起重要作用，包括DNA修复、染色质调节、转录、凋亡、炎症和衰老。SSB和复制毒物也触发ATR-Chk1 DNA损伤反应(DDR)途径。Chk1被ATR磷酸化是S阶段检查点的一个关键事件。ATR可在S期检查点被PAR化，PARP抑制剂可过度激活Chk1，导致S期停滞。这些观察结果表明，PAR化在ATR-Chk1介导的S阶段检查点控制中发挥了作用。我们最近在Chk1中发现了一个新的PAR结合基序(PBR)，并发现了PAR代谢和细胞周期进程控制之间的相互作用。当PAR的长链刺激Chk1激酶活性时，短链PAR抑制这种刺激，这意味着PAR的动态平衡对S阶段的检查点进行了动态微调。我们还发现，PBR突变使BRCA1突变的癌细胞对细胞死亡敏感，这表明对乳腺癌细胞的协同杀伤。然而，PAR在体内与Chk1结合的生物学意义和分子机制尚不清楚。因此，我们建议开展以下研究：(1)明确PAR和Chk1在ATR-Chk1途径中的相互作用，以响应SSB和复制。(2)建立PBR敲击小鼠模型，研究CHK1的PBR基序在细胞和小鼠的存活、胚胎和出生后发育以及组织内稳态方面的生物学意义。我们将改造携带突变的PBR-Chk1(PbRC/A-Chk1)的敲入小鼠，利用遗传学、细胞和分子工具剖析PAR与Chk1结合的生物学功能。我们将分析PbRC/A-Chk1突变小鼠的一般和神经发育，以检查这些小鼠在胚胎发育或出生后生活中是否表现出高水平的复制应激，或者是否表现出加速衰老，这可能模仿ATR亚型小鼠的Seckel表型。这些研究将描述ATR-Chk1和PARP1/PAR网络在体内处理DNA损伤信号中的生物学功能。探讨PAR代谢在ATR-Chk1激活中的特殊作用，可能为人类恶性肿瘤的治疗提供新的治疗策略。

Single-stranded DNA breaks (SSBs) and replication stress activate PARP1 that catalyses the formation of poly(ADP-ribose) (PAR) polymers as well as the PARylation of target proteins. PAR has been hypothesised to function as a signal to other cellular targets, including repair proteins. PARylation and PARP1 have been shown to be important for multiple cellular processes, including DNA repair, chromatin modulation, transcription, apoptosis, inflammation and ageing. SSBs and replication poison also trigger the ATR-Chk1 DNA damage response (DDR) pathway. Chk1 phosphorylation by ATR is a key event in the S-phase checkpoint. ATR can be PARylated in the S-phase checkpoint and the PARP inhibitor can over-activate Chk1 leading to the S-phase arrest. These observations suggest a role of PARylation in the ATR-Chk1-mediated S-phase checkpoint control. We recently identified a novel PAR-binding motif (PbR) in Chk1 and found interplay between the PAR metabolism and cell cycle progression control. While long chains of PAR stimulate the Chk1 kinase activity, short chains of PAR repress this stimulation, implying a dynamic fine-tuning of the S-phase checkpoint by PAR homeostasis. We also found that a PbR mutation sensitises BRCA1 mutant cancer cells to cell death, which indicates a synergistic killing of breast cancer cells. However, the biological significance and the molecular mechanisms of PAR binding to Chk1 in vivo are largely unknown. Thus, we propose to carry out the following studies:(1) To define the interactive roles of PAR and Chk1 in the ATR-Chk1 pathway in response to SSB and replication. (2) To generate the PbR knockin mouse model to investigate the biological significance of the PbR motif of Chk1 in cells and mice for viability, embryonic and postnatal development and tissue homeostasis. We will engineer knock-in mice that carry the mutant PbR-Chk1 (PbRC/A-Chk1) to dissect the biological function of PAR binding to Chk1 using genetics, cellular and molecular tools. We will analyse the general and neuro-development of the PbRC/A-Chk1 mutant mice to examine whether these mice exhibit a high level of replicative stress during embryogenesis or postnatal life, or accelerated ageing, which may mimic the Seckel phenotype of ATR hypomorphic mice. The proposed studies will delineate the biological function of the network of ATR-Chk1 and PARP1/PAR in handling DNA damage signals in vivo. Exploring the specific function of the PAR metabolism in the ATR-Chk1 activation may lead to new therapeutic strategies for the treatment of human malignancy.