Unveiling the mechanism for Spartan-mediated DNA lesion bypass and the implications for breast cancer treatment

 描述（由申请方提供）：在DNA复制期间，UV暴露产生的损伤导致复制叉停滞。为了通过这些损伤合成DNA，复制聚合酶（Polδ）必须与特殊聚合酶（Polη）交换，后者可以掺入损伤对面的核苷酸。这个过程被称为translsion Synthesis（TLS）。聚合酶转换的机制仍不清楚。增殖细胞核抗原（PCNA）的单泛素化是该过程有效发生所必需的。我们的实验室先前已经鉴定了一种名为Spartan的蛋白质，它与泛素化的PCNA结合，并促进Polη向DNA损伤的募集。目前尚不清楚Spartan如何介导聚合酶开关。我的初步数据表明，在紫外线照射斯巴达调节复制聚合酶，POLD 4的辅助亚基的稳定性。TLS在癌症中的重要性由常染色体隐性疾病着色性干皮病变体（XPV）例证，其中Polη在功能上无活性。XPV患者对紫外线暴露的敏感性增加，导致皮肤癌的易感性急剧增加。检索基因组学在线数据库桑格COSMIC，发现10%的乳腺癌组织检测有SPRTN/C1 orf 124扩增，30%有过表达。这表明斯巴达失调可能会影响乳腺癌的发展，使细胞能够处理伴随基因组不稳定性的复制压力，这是癌症的标志。该提案的具体目的如下：（1）确定Spartan是否通过促进POLD 4降解来调节Polδ-to-Pol η开关;（2）阐明Spartan如何通过其SprT结构域调节TLS;（3）确定乳腺癌中SPRTN的扩增是否为治疗提供了机会。为了解决这些目标，将测试破坏各种功能结构域的Spartan突变体促进POLD 4从PCNA解离以及Polη与PCNA缔合的能力。将分别通过co-IP和免疫荧光法评估这些相互作用和共定位。将使用体外测定来确定SprT结构域是否具有蛋白酶活性并切割POLD 4。此外，将生成Spartan诱导型稳定细胞系。这将用于揭示Spartan过表达对未转化乳腺上皮细胞对外源性DNA损伤或内在基因组不稳定性的耐受性的影响。还将评估shRNA介导的Spartan敲低对乳腺癌细胞活力和增殖的影响。拟议方案的短期贡献 该研究将阐明Spartan在TLS（一种癌症相关通路）中的作用机制。长期的贡献可能是开发消除癌症的新药。

 DESCRIPTION (provided by applicant): During DNA replication, lesions generated by UV exposure result in replication fork stalling. To synthesize DNA past these lesions, replicative polymerases (Polδ) must be exchanged with special polymerases (Polη), which can incorporate nucleotides opposite of lesions. This process is named Translesion Synthesis (TLS). The mechanism of polymerase switching remains unclear. Monoubiquitination of Proliferating Cell Nuclear Antigen (PCNA) is required for this process to occur efficiently. Our lab has previously identified a protein named Spartan that binds to ubiquitinated PCNA and promotes the recruitment of Polη to DNA lesions. It is currently unclear how Spartan mediates the polymerase switch. My preliminary data suggests that during UV exposure Spartan regulates the stability of an accessory subunit of the replicative polymerase, POLD4. The importance of TLS in cancer is exemplified by the autosomal recessive disease Xeroderma Pigmentosum Variant (XPV) in which Polη is functionally inactive. XPV patients have an increased sensitivity to UV exposure that results in a dramatic increased susceptibility to skin cancer. Searching the genomics online database Sanger COSMIC, revealed that 10% of breast cancer tissues tested had SPRTN/C1orf124 amplifications and 30% had overexpression. This suggests that Spartan dysregulation might influence breast cancer development by enabling cells to handle replication stress that accompanies genome instability, a hallmark of cancer. The specific aims of this proposal are as follows: (1) To determine whether Spartan regulates the Polδ-to-Polη switch by promoting POLD4 degradation; (2) to elucidate how Spartan regulates TLS through its SprT domain; (3) to determine whether amplification of SPRTN in breast cancer offers an opportunity for therapy. To address these aims, Spartan mutants that disrupt the various functional domains will be tested for their ability to promote POLD4 dissociation from PCNA, and Polη association with PCNA. These interactions and co-localizations will be assessed by co-IP and Immunofluorescence, respectively. In vitro assays will be used to determine whether the SprT domain harbors protease activity and cleaves POLD4. Also, a Spartan inducible stable cell line will be generated. This will be used to reveal the impact of Spartan overexpression on the tolerance of untransformed mammary epithelial cells to extrinsic DNA damage or intrinsic genomic instability. The influence of shRNA mediated Spartan knock-down on breast cancer cell viability and proliferation will also be assessed. The short-term contribution of the proposed research will be the illumination of the mechanism of Spartan in TLS, a cancer relevant pathway. The long-term contribution could be the development of new drugs for eliminating cancer.