Non-canonical cGAS signaling in DNA damage response

摘要 脊椎动物的先天免疫依赖于一组复杂的胞浆模式识别受体(PRR)来 检测病原体衍生的、进化上保守的分子，如DNA。细胞内DNA的传感技术 环-GMP-AMP合成酶(CGAS)激活环鸟苷单磷酸的酶促合成。 腺苷一磷酸(CGAMP)，环二核苷酸(CDN)的第二信使。通过ITS发送cGAMP信号 高亲和力受体蛋白刺痛，随后招募坦克结合蛋白1(TBK1)和干扰素 干扰素调节因子3(IRF3)刺激I型IFN的诱导。尽管最初被标识为 外源DNA的胞浆传感器，cGAS也被来自于 胞浆或微核中的基因组DNA受损。 最近，多项趋同研究强调了cGAS在DNA损伤中的重要性。 炎症反应及其对细胞衰老、肿瘤发生和转移的影响。没什么, 然而，我们知道在这些环境中激活cGAS是否直接有助于维护 基因组的完整性。我们实验室最近的研究发现，cGAS/cGAMP信号触发 DNA损伤反应(DDR)，独立于其特征明确的I型干扰素途径。这些 研究表明，cGAMP诱导的DDR激活细胞周期检查点反应，导致G1期停滞 和随后抑制双链DNA断裂(DSB)的同源定向修复(HDR) CRISPR/Cas9编辑的小鼠胚胎以及人和鼠细胞。有趣的是，cGAMP诱导的DDR 在缺乏干扰素的无脊椎动物物种(牡蛎和海葵)中也有表现。 免疫系统，提示cGAMP的DNA损伤监测机制早于其更好的 已知的干扰素介导的免疫功能。 本文提出的研究旨在通过阐明血管紧张素转换酶的分子机制来推进这些新发现。 CGAS/cGAMP通过三个主题整合但又独立的目的诱导DDR：(1)解密 CGAMP诱导ATM激活的关键信号通路；(2)定义分子 CGAS-cGAMP抑制HDR的机制；(3)阐明cGAMP是否诱导DDR 增强cGAS启动的先天免疫力。总而言之，这项工作将阐明 CGAMP诱导顶端DDR信号通路ATM激活的分子和生化基础，以及 加深对cGAMP诱导的DDR信号与传统免疫的关系的理解 CGAS的功能。

SUMMARY The vertebrate innate immunity relies upon a complex set of cytosolic pattern recognition receptors (PRR) to detect pathogen-derived, evolutionarily conserved molecules such as DNA. The sensing of cytosolic DNA by cyclic-GMP-AMP synthase (cGAS) activates the enzymatic synthesis of cyclic guanosine monophosphate- adenosine monophosphate (cGAMP), a cyclic dinucleotide (CDN) second messenger. cGAMP signals via its high affinity receptor protein STING, which subsequently recruits TANK-binding kinase 1 (TBK1) and interferon (IFN) regulatory factor 3 (IRF3) to stimulate the induction of type I IFNs. Although originally identified as a cytosolic sensor of foreign DNA, cGAS is also recruited to and activated by fragments of chromatin from damaged genomic DNA in the cytosol or micronuclei. Multiple convergent studies have recently highlighted the significance of cGAS in the DNA damage–induced inflammatory response and its implications for cellular senescence, tumorigenesis, and metastasis. Nothing, however, is known about whether cGAS activation in these contexts directly contributes to the maintenance of genome integrity. Recent studies in our laboratory have discovered that cGAS/cGAMP signaling triggers DNA damage response (DDR), independently of its well-characterized type I interferon pathway. These studies revealed that cGAMP-induced DDR activates cell cycle checkpoint responses that lead to G1 arrest and subsequent suppression of homology directed repair (HDR) of double-strand DNA breaks (DSB) in CRISPR/Cas9-edited mouse embryos and human and mouse cells. Interestingly, the cGAMP-induced DDR was also demonstrable in invertebrate species (oysters and starlet sea anemone) lacking interferon-based immune system, suggesting that the DNA damage surveillance mechanism of cGAMP predates its more well- known IFN-mediated immune function. The studies proposed here aim to advance these novel findings by elucidating the molecular mechanism of cGAS/cGAMP-induced DDR induction via three thematically integrated, yet independent Aims: (1) Decipher the critical signaling pathways involved in cGAMP-induced ATM activation; (2) Define the molecular mechanism of cGAS-cGAMP-induced suppression of HDR; and (3) Elucidate whether cGAMP-induced DDR potentiates the cGAS-initiated innate immunity. In summary, this work will illuminate novel aspects of the molecular and biochemical basis of cGAMP-induced activation of the apical DDR signaling kinase ATM, and increase understanding of the relationship between cGAMP-induced DDR signaling and the traditional immune function of cGAS.