Non-canonical cGAS signaling in DNA damage response

总结 脊椎动物先天免疫依赖于一组复杂的胞质模式识别受体（PRR）， 检测病原体衍生的、进化上保守的分子，如DNA。细胞质DNA的传感 环GMP-AMP合酶（cGAS）激活环鸟苷一磷酸的酶促合成- 腺苷一磷酸（cGAMP），一种环二核苷酸（CDN）第二信使。cGAMP信号通过其 高亲和力受体蛋白STING，其随后募集TANK结合激酶1（TBK 1）和干扰素 (IFN)调节因子3（IRF 3）刺激I型IFN的诱导。虽然最初被确定为一个 作为外源DNA的胞质传感器，cGAS也被来自 细胞质或微核中受损的基因组DNA。 最近，多项聚合研究强调了cGAS在DNA损伤诱导的细胞凋亡中的重要性。 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，已知在这些情况下cGAS激活是否直接有助于维持 基因组的完整性。我们实验室最近的研究发现，cGAS/cGAMP信号转导触发了 DNA损伤反应（DDR），独立于其充分表征的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期阻滞 以及随后抑制双链DNA断裂（DSB）的同源定向修复（HDR）， CRISPR/Cas9编辑的小鼠胚胎以及人类和小鼠细胞。有趣的是，cGAMP诱导的DDR 在缺乏干扰素的无脊椎动物物种（牡蛎和小海葵）中也是明显的 免疫系统，表明cGAMP的DNA损伤监测机制早于其更好的- 已知IFN介导的免疫功能。 本文提出的研究旨在通过阐明这些新发现的分子机制， cGAS/cGAMP通过三个主题集成但独立的目标诱导DDR： cGAMP诱导ATM活化的关键信号通路;（2）确定ATM活化的分子机制。 cGAS-cGAMP诱导的HDR抑制的机制;和（3）阐明cGAMP诱导的DDR是否 增强cGAS启动的先天免疫。总之，这项工作将阐明新的方面， cGAMP诱导的顶端DDR信号激酶ATM活化的分子和生物化学基础，以及 增加对cGAMP诱导的DDR信号传导与传统免疫系统之间关系的理解。 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.