Post-translational regulation of the uracil DNA glycosylase

 说明(申请人提供)：胸苷酸合酶抑制剂是一类广泛用于治疗各种癌症的药物。这些化合物的作用之一是增加新合成的基因组DNA中尿嘧啶碱基的水平。过量的尿嘧啶掺入DNA会对细胞产生毒性，这被认为是胸苷合成酶抑制剂靶向快速分裂的癌细胞的原因之一。启动碱基切除修复途径以纠正基因组DNA中这些尿嘧啶损伤的主要DNA修复酶称为核尿嘧啶DNA糖基酶(UNG2)。我们提出了UNG2的尿嘧啶清除活性与5-氟尿嘧啶的疗效之间的关系，5-氟尿嘧啶是一种典型的胸苷合成酶抑制剂。UNG2的活性在一定程度上受到它在细胞核内的定位以及它与DNA损伤部位的可及性的调节。UNG2与增殖细胞核抗原(PCNA)和复制蛋白A(RPA)的相互作用可以影响其定位，特别是对DNA复制叉处的定位；因此，我们推测在5-氟尿嘧啶治疗期间，它从新合成的DNA中去除尿嘧啶的能力依赖于这些蛋白质-蛋白质相互作用。增殖细胞核抗原和RPA结合域中的特定UNG2残基可以翻译后修饰，我们假设这些翻译后修饰(PTM)影响UNG2与这些蛋白质的相互作用。因此，UNG2的翻译后修饰被认为是通过影响蛋白质-蛋白质相互作用以及随后从DNA中去除尿嘧啶来调节癌细胞对5-氟尿嘧啶毒性的敏感性。为了验证我们的假设，我们将使用最先进的蛋白质半合成方法来产生在特定残基掺入PTM的UNG2变体。翻译后修饰的UNG2变异体将用于生化分析，以量化它们的活性及其与增殖细胞核抗原和RPA的亲和力。还将确定调控UNG2与增殖细胞核抗原和RPA相互作用的结构基础。为了证实PTM影响蛋白质-蛋白质相互作用和UNG2在细胞核中的定位，我们将使用显微注射半合成UNG2变体的活细胞荧光成像。最后，5-氟尿嘧啶的疗效将在转染了改变了增殖细胞核抗原和/或RPA亲和力的UNG2突变体的细胞中进行检验。总而言之，这一跨学科项目的结果将解决一组特定的UNG2 PTM对5-氟尿嘧啶和其他胸苷合成酶抑制剂疗效的贡献。

 DESCRIPTION (provided by applicant): Thymidylate synthase inhibitors are a class of drugs that are widely used for the treatment of various cancers. One effect of these compounds is to increase the levels of uracil bases that are incorporated into newly synthesized genomic DNA. Excessive uracil incorporation into DNA can become toxic to cells, and this is believed to contribute to the ability of thymidylate synthase inhibitors to target rapidly dividing cancer cell. The primary DNA repair enzyme that initiates the base excision repair pathway to correct these uracil lesions in genomic DNA is called the nuclear uracil DNA glycosylase (UNG2). We propose a relationship between the uracil excision activity of UNG2 and the efficacy of 5-fluorouracil, a prototypical thymidylate synthase inhibitor. The activity of UNG2 is regulated in part by its localization within the nucleus and its accessibility to sites of DNA damage. Interactions of UNG2 with proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) can affect its localization, specifically to the DNA replication fork; therefore, we reason that its ability to excise uracil from newly synthesized DNA during 5-fluorouracil treatment is als dependent on these protein- protein interactions. Specific UNG2 residues within the PCNA and RPA binding domains can be post- translationally modified, and we hypothesize that these post-translational modifications (PTMs) affect UNG2 interactions with these proteins. Post-translational modification of UNG2 is therefore hypothesized to modulate the susceptibility of cancer cells to 5-fluorouracil toxicity by affecting protein-protein interactions and subsequently removal of uracil from DNA. To test our hypotheses, we will use state-of-the-art protein semi- synthesis methods to generate UNG2 variants that have PTMs incorporated at specific residues. The post- translationally modified UNG2 variants will be used in biochemical assays that quantify their activity and their affinities for PCNA and RPA. The structural basis for modulating UNG2 interactions with PCNA and RPA will also be determined. To confirm that PTMs affect protein-protein interactions and the localization of UNG2 in the nucleus, we will use live cell fluorescence imaging of microinjected semi-synthetic UNG2 variants. Finally, the efficacy of 5-fluorouracil will be examined in cells that are transfected with UNG2 mutants that have altered affinities for PCNA and/or RPA. Together, the results of this interdisciplinary projec will address the contribution of a specific set of UNG2 PTMs towards the efficacy of 5-fluorouracil and other thymidylate synthase inhibitors.