Post-translational regulation of the uracil DNA glycosylase

 描述（由申请人提供）：胸苷酸合成酶抑制剂是一类广泛用于治疗各种癌症的药物。这些化合物的一个作用是增加掺入新合成的基因组DNA中的尿嘧啶碱基的水平。过量的尿嘧啶掺入DNA中会对细胞产生毒性，据信这有助于胸苷酸合成酶抑制剂靶向快速分裂的癌细胞的能力。启动碱基切除修复途径以纠正基因组DNA中的这些尿嘧啶损伤的主要DNA修复酶被称为核尿嘧啶DNA糖基化酶（UNG 2）。我们提出了UNG 2的尿嘧啶切除活性与5-氟尿嘧啶（一种原型胸苷酸合成酶抑制剂）的疗效之间的关系。UNG 2的活性部分受其在细胞核内的定位及其对DNA损伤位点的可及性调节。UNG 2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DNA复制叉;因此，我们推断其在5-氟尿嘧啶治疗期间从新合成的DNA中切除尿嘧啶的能力也依赖于这些蛋白质-蛋白质相互作用。PCNA和RPA结合结构域内的特定UNG 2残基可以被翻译后修饰，并且我们假设这些翻译后修饰（PTM）影响UNG 2与这些蛋白质的相互作用。因此，假设UNG 2的翻译后修饰通过影响蛋白质-蛋白质相互作用和随后从DNA中去除尿嘧啶来调节癌细胞对5-氟尿嘧啶毒性的易感性。为了测试我们的假设，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处掺入PTM的UNG 2变体。后修饰的UNG 2变体将用于定量其活性及其对PCNA和RPA的亲和力的生物化学测定。还将确定调节UNG 2与PCNA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UNG 2在细胞核中的定位，我们将使用显微注射的半合成UNG 2变体的活细胞荧光成像。最后，将在用对PCNA和/或RPA具有改变的亲和力的UNG 2突变体转染的细胞中检查5-氟尿嘧啶的功效。总之，这个跨学科项目的结果将解决一组特定的UNG 2 PTM对5-氟尿嘧啶和其他胸苷酸合成酶抑制剂的疗效的贡献。

 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.