PAR binding to Chk1 regulates development and tissue homeostasis

单链DNA断裂（SSB）和复制应激激活PARP 1，PARP 1催化聚（ADP-核糖）（PAR）聚合物的形成以及靶蛋白的PAR化。PAR已被假设为作为其他细胞靶点的信号，包括修复蛋白。PAR化和PARP 1已被证明对多种细胞过程是重要的，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-Chk 1 DNA损伤反应（DDR）途径。ATR对Chk 1的磷酸化是S期检查点的关键事件。ATR可以在S期检查点被PAR化，PARP抑制剂可以过度激活Chk 1，导致S期阻滞。这些观察结果表明PAR化在ATR-Chk 1介导的S期检查点控制中的作用。我们最近发现了一个新的PAR结合基序（PbR）在Chk 1和PAR代谢和细胞周期进程控制之间的相互作用。虽然PAR的长链刺激Chk 1激酶活性，但PAR的短链抑制这种刺激，这意味着通过PAR稳态对S期检查点进行动态微调。我们还发现，PbR突变使BRCA 1突变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与Chk 1结合的生物学意义和分子机制在很大程度上是未知的。因此，我们建议进行以下研究：（1） 明确PAR和Chk 1在ATR-Chk 1通路中对SSB和复制的相互作用。（二） 建立PbR基因敲入小鼠模型，研究Chk 1基因PbR基序在细胞和小鼠生存能力、胚胎和出生后发育以及组织稳态中的生物学意义。我们将利用遗传学、细胞学和分子学工具，设计携带突变型PbR-Chk 1（PbRC/A-Chk 1）的基因敲入小鼠，以剖析PAR与Chk 1结合的生物学功能。我们将分析PbRC/A-Chk 1突变小鼠的一般和神经发育，以检查这些小鼠在胚胎发生或出生后生活或加速衰老期间是否表现出高水平的复制应激，这可能模拟ATR亚型小鼠的Seckel表型。本研究将探讨ATR-Chk 1和PARP 1/PAR网络在体内处理DNA损伤信号中的生物学功能。探索PAR代谢在ATR-Chk 1激活中的特定功能可能会为人类恶性肿瘤的治疗带来新的治疗策略。

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.