DNA聚合酶eta O-GlcNAc糖基化修饰对其功能的调控研究

Translesion DNA synthesis (TLS) plays an important role in genome mutagenesis and chemotherapeutic drug resistance. How TLS pathway is regulated remains a hot topic in the field of DNA damage response. DNA polymerase η (Polη) can bypass UV-induced CPD lesions correctly and efficiently, whose deficiency leads to skin tumors. Polη also facilitates TLS past cisplatin-induced DNA lesions, which is closely related to chemotherapeutic drug resistance in ovarian tumors. In our preliminary studies, we found that human Polη interacts with O-GlcNAc transferase (OGT) and is subject to O-GlcNAcylation. Mutation of several potential residues in Polη to reduce its O-GlcNAcylation affects genome mutagenesis and cellular resistance to UV irradiation and cisplatin treatment. By integrating multiple cellular, biochemical and molecular techniques, this project aims at exploring the effects of Polη O-GlcNAcylation on its foci formation and replication across damaged DNA as well as cellular resistance in response to UV irradiation and cisplatin treatment, and determining the role(s) of Polη O-GlcNAcylation in cisplatin-induced enrichment of ovarian tumor stem cells and chemotherapeutic drug resistance of ovarian tumor. Unveiling of the molecular mechanisms of Polη O-GlcNAcylation on TLS regulation will provide important insights into the understanding of carcinogenesis and prevention of chemotherapeutic drug resistance.

跨损伤DNA合成（TLS）通路在基因组变异和肿瘤耐药性产生中发挥重要作用，关于其调控机制研究一直是DNA损伤应答领域一个热点。TLS聚合酶Polη能正确复制紫外辐射诱发的CPD加合物，缺失Polη导致皮肤癌发生率显著升高。Polη还能复制顺铂诱发的损伤，增加卵巢癌化疗耐药性。在前期研究中，申请人发现O-GlcNAc糖基化酶OGT与Polη结合并介导Polη发生O-GlcNAc糖基化修饰，降低该修饰干扰细胞的TLS活性。本项目将整合多种分子和细胞生物学技术、结合小鼠模型，深入研究Polη O-GlcNAc糖基化修饰对其在损伤位点募集、DNA复制和基因组突变的影响，阐明该修饰调控TLS通路的作用机制；探讨该修饰对顺铂诱发的卵巢癌干细胞富集和卵巢癌治疗的影响，阐明其与卵巢癌化疗耐药的关联，为深入理解癌症发生和降低化疗耐药性提供重要理论基础。

跨损伤DNA合成（TLS）通路在基因组变异和肿瘤耐药性产生中发挥重要作用，关于其调控机制研究一直是DNA损伤应答领域一个热点。TLS聚合酶Polη能正确复制紫外辐射诱发的CPD加合物，缺失Polη导致皮肤癌发生率显著升高。Polη还能复制顺铂诱发的损伤，增加肿瘤化疗耐药性。我们发现Polη与O-GlcNAc糖基化酶OGT结合并发生O-GlcNAc糖基化修饰。整合多种分子和细胞生物学技术，我们发现Polη O-GlcNAc糖基化修饰主要发生在T457位点，干扰其O-GlcNAc糖基化修饰并不影响Polη到UV损伤位点的招募和复制CPD能力，而是影响Polη发挥TLS功能后从UV和顺铂损伤位点的及时移除。机制上，发现Polη T457位点O-GlcNAc糖基化修饰促进邻近位点K462发生E3 CRL4CDT2介导的K48链多泛素化修饰，进而通过p97复合物介导从染色质上及时移除。干扰Polη O-GlcNAc糖基化修饰影响细胞在UV和顺铂暴露后的损伤应答、复制叉移动速度，影响顺铂诱发的肿瘤干细胞富集和细胞对药物敏感性。这些发现有助于我们深入理解TLS通路的调控和Polη在受阻复制叉的招募/移除，为预防卵巢癌等发生以及以TLS通路作为肿瘤耐药性药物靶标提供了重要的理论基础。受该基金的资助，目前已经发表SCI 文章11 篇，包括Nature commun (2017)（得到F1000推荐）、Nucleic Acids Res (2017a,b，2018)、PloS Biol (2016)等。