跨损伤合成聚合酶polη泛素化修饰的功能研究

Translesion DNA synthesis (TLS) is an important DNA damage tolerance mechanism, plays key roles in genomic stability and development of cancer drug resistance, which is also a hot topic in DNA repair research. TLS polymerases Polη plays a key role in TLS process，which can bypass the DNA lesion induced by UV and cisplatin and is closely related with the ccurrence of skin cancer and chemotherapeutic drug resistance. Ubiquitination modification can regulate the stability, interaction with other repair proteins, recuitment and remove at stalling repelicaiton folks of Polη. At present, the function of many ubiqitination sites on Polη are not clear and few ubiquitination modification enzymes are reported. We found two new multi-ubiquitination modification enzymes of Polη in the previous work, named Polη-E3-1 and Polη-DUB-1，and that the knock-down of Polη-E3-1 has an influence on the recuitment of Polη under UV radiation. In this study, we will use various molecular and cellular biotechniques to expore the regulation roles of these two ubiquitination modification enzymes on Polη ubiquitin level and other cellular functions. We believe that the results will help understand the function of translesion bypass polymerases and has an important significance on regulating research between TLS and other DNA repair pathways.

跨损伤合成(TLS)是一种重要DNA损伤耐受机制，在基因组稳定性和肿瘤抗药性发展中发挥关键作用，也是DNA损失修复研究领域的热点问题。TLS聚合酶Polη在TLS过程中发挥重要作用，它能正确复制紫外辐射和顺铂诱发的DNA损伤，与皮肤癌发生和肿瘤耐药密切相关。泛素化修饰影响Polη在细胞内的稳定性、与其他蛋白相互作用、在停顿复制叉的招募和解离过程。目前，Polη上多个泛素化位点功能尚未清楚，相应的泛素化修饰酶也少有报道。我们前期实验发现了2个新的Polη多泛素化修饰酶Polη-E3-1和Polη-DUB-1，并且敲低Polη-E3-1表达影响UV损伤条件下Polη的招募。在本项目中，我们将用多种分子细胞生物学技术，深入研究这两种泛素化修饰酶对Polη泛素化的调控作用和细胞内功能的影响。研究结果将有助于进一步了解Polη的功能，对TLS和其他DNA修复通路的相互调控关系研究也具有重意义。

Polη能够正确复制紫外线UV和顺铂CDDP诱发的DNA损伤，与皮肤癌的发生和多种肿瘤药物的耐药性密切相关。目前，关于泛素化修饰调控Polη的功能机制所知甚少。我们发现E3连接酶TRIM21与Polη存在相互作用，能够催化介导Polη的K48和K63连接的多泛素化修饰，促进细胞内Polη的稳定性。用siRNA方法敲低TRIM21影响了UV损伤后PCNA单泛素化（PCNA-mUb）水平和染色质结合的Polη量，对RPA和RAD18的招募没有影响。我们还发现敲低TRIM21表达，影响了UV损伤后Polη的招募和Polη跨过环丁烷嘧啶二聚体（CPD）损伤的聚合酶活性，导致在细胞对UV损伤敏感性增强。这些发现有助于我们了解泛素化修饰对Polη的影响，为研究TLS调控机制和化疗药物耐药性具有重要意义。受该基金的资助，目前已经发表SCI文章2篇，包括Nucleic Acids Res (2018)和Biochim Biophys Acta Proteins Proteom (2018)等。