【作者】 张杰；

【导师】 赵国平；

【作者基本信息】 中国科学技术大学 ， 生物物理学， 2025， 博士

【摘要】 放射治疗和化疗仍然是当前多种恶性肿瘤的重要治疗手段,其核心机制在于通过电离辐射或化学药物诱导肿瘤细胞发生DNA损伤,此类损伤可激活细胞内不同信号网络,导致增殖阻滞及程序性死亡等生物学效应,从而抑制了肿瘤的发展并促进了肿瘤的根除。然而,肿瘤细胞通过同源重组修复(HR)通路异常活化及非同源末端连接(NHEJ)的机制失调,显著增强了DNA修复保真度,常表现出对放/化疗的抗性,严重削弱干预效果。因此,解析DNA损伤应答分子调控网络以提升常规治疗方案的肿瘤细胞杀伤效率,成为亟待解决的关键科学问题。作为复制蛋白A(RPA)复合体的关键功能亚基,RPA2通过稳定结合单链DNA(ssDNA)并协同调控修复因子募集,在HR介导的DNA损伤修复通路中具有着不可替代的生物学功能。然而,关于RPA2与ssDNA的动态结合及其如何精密调控修复蛋白复合物的有序组装,仍是DNA损伤领域的一大科学盲区。为此,本研究采用免疫共沉淀联合液-相色谱/串联质谱(Co-IP-LC-MS/MS)联用技术进行互作蛋白组学分析,最终鉴定出E3泛素连接酶TRIM21作为RPA2的新型互作效应分子。TRIM21作为TRIM蛋白家族的重要成员,以往研究多聚焦于其免疫调控功能,而其在DNA损伤修复及肿瘤进程中的角色尚待挖掘。基于一系列的体外以及体内实验,本研究首次阐明了TRIM21在HR修复起始阶段发挥特异性调控功能的分子机制。在未受损的状态下,TRIM21与RPA2直接相互作用。该互作不仅促进RPA2上K63连接的泛素化修饰,还显著抑制其DNA结合域(DBD-D)内K6连接的泛素化。当DNA损伤发生后,TRIM21凭借其PRYSPRY结构域被迅速招募至损伤位点;与此同时,RPA2的这种K63连接的泛素化修饰增强了其与ssDNA的结合能力,进而稳定损伤位点的结构。这一过程有效促进了ATRIP和RAD51等修复因子的募集,保障HR修复的高效推进。进一步研究发现,ATR激酶在TRIM21调控中具有关键作用。ATR通过磷酸化TRIM21的第41位丝氨酸(Ser41)残基,触发其与RPA2的解离过程。该翻译后修饰不仅动态调控了RPA2的泛素化(由K63连接向K6连接模式)的转换,更重要的是确保了RPA2-ATRIP复合物的稳定形成,并推动修复进程的顺利过渡。本研究通过系统性分析TRIM21协同E3泛素连接酶活性与ATR激酶介导的磷酸化修饰网络,详细阐明了TRIM21调控RPA2与ssDNA结合及修复因子特异性募集的分子机制。此外,TRIM21在多种肿瘤中呈现高表达,并显著影响肿瘤细胞对放/化疗的敏感性。本研究不仅深化了对HR修复分子机制的理解,还揭示了TRIM21在肿瘤抗性中的潜在作用,凸显其作为癌症治疗靶点的应用前景,为开发新型治疗策略提供了理论依据。更多还原

【Abstract】 Radiotherapy and chemotherapy remain critical treatment modalities for various malignant tumors.Their core mechanism lies in inducing DNA damage in tumor cells through ionizing radiation or chemotherapeutic agents.Such damage activates intracellular signaling networks,triggering biological effects such as proliferative arrest and programmed cell death,thereby inhibiting tumor progression and promoting eradication.However,tumor cells significantly enhance DNA repair fidelity through abnormal activation of the homologous recombination repair(HR)pathway and dysregulation of the non-homologous end joining(NHEJ)mechanism,frequently exhibiting resistance to radiotherapy and chemotherapy.This severely compromises therapeutic efficacy.Consequently,deciphering the molecular regulatory network of DNA damage response to improve the tumor cell-killing efficiency of conventional treatment regimens has emerged as a critical scientific challenge requiring urgent resolution.As a key functional subunit of the replication protein A(RPA)complex,RPA2has an irreplaceable biological role in the HR-mediated DNA damage repair pathway by stably binding to single-stranded DNA(ssDNA)and synergistically regulating repair factor recruitment.However,the dynamic binding of RPA2 to ssDNA and its precise regulatory role in orchestrating the ordered assembly of repair protein complexes remain a critical gap in our understanding of DNA damage response.To address this,our study employed co-immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry(Co-IP-LC-MS/MS)for interactome profiling,ultimately identifying the E3 ubiquitin ligase TRIM21 as a novel interacting effector of RPA2.As an important member of the TRIM protein family,TRIM21 has been predominantly studied for its immune-regulatory functions,while its roles in DNA damage repair and tumor progression remain underexplored.Through a series of in vitro and in vivo experiments,this study elucidated for the first time the molecular mechanism by which TRIM21 specifically regulates the initiation phase of HR repair.In an undamaged state,TRIM21 directly interacts with RPA2,promoting K63-linked ubiquitination while markedly suppressing K6-linked ubiquitination within RPA2’s DNA-binding domain(DBD-D).Upon DNA damage,TRIM21,via its PRYSPRY domain,is rapidly recruited to damage sites,while K63-linked ubiquitination of RPA2 enhance its binding affinity to ssDNA,thereby stabilizing the structural integrity of the damaged region.This process effectively facilitating the recruitment of repair factors such as ATRIP and RAD51,thereby ensuring efficient HR repair progression.Further investigation revealed the pivotal role of ATR kinase in TRIM21regulation.ATR phosphorylates the Serine 41 residue(Ser41)of TRIM21,triggering its dissociation from RPA2.This post-translational modification not only dynamically regulates the transition of RPA2 ubiquitination(from K63-linked to K6-linked chain topology)but,more critically,ensures the stable assembly of the RPA2-ATRIP complex and facilitates the seamless progression of the DNA repair process.This study systematically analyzed the coordinated E3 ubiquitin ligase activity of TRIM21 and the ATR kinase-mediated phosphorylation regulatory network,providing a detailed elucidation of the molecular mechanisms through which TRIM21 regulates RPA2 binding to ssDNA and orchestrates the specific recruitment of DNA repair factors.Moreover,TRIM21 exhibits elevated expression in various tumors and significantly modulates tumor cell sensitivity to radio/chemotherapy.This research not only deepens the understanding of HR repair’s molecular mechanisms but also highlights TRIM21’s potential role in tumor resistance,underscoring its promise as a therapeutic target for cancer and providing a theoretical foundation for developing novel treatment strategies.更多还原