Role of nuclear chaperones in genomic instability and carcinogenesis

摘要 DNA中的损伤、断裂和错误是基因组不稳定的驱动因素，导致各种癌症。这个 DNA损伤响应(DDR)是对DNA中的断裂做出反应并利用阵列的信号级联 DNA修复因子来纠正错误并保持基因组的完整性。细胞对DDR的反应包括 细胞周期和信号过程的调节，可触发DNA修复或细胞程序性死亡。 在DDR中起作用的蛋白质被穿梭到细胞核中，以回应DNA损伤。我的博士论文工作 提出的目标围绕着理解调节核质的机制。 DDR蛋白的定位。该提案的F99部分侧重于对复制蛋白A的调控 (RPA)通过伴侣样蛋白。RPA是一种基本的单链DNA(SsDNA)结合DDR因子， 调节DNA代谢的方方面面，包括DNA复制、修复和重组。RPA是 转运到核中，识别和结合单链DNA，并通过与三个以上的 十几个RPA相互作用蛋白(RIPs)。如何在中的单元中防止虚假RPA-RIP交互 单链DNA的缺失一直是个谜。我发现了Rtt105(Ty1的调节子 转座105)是一种伴侣样蛋白，通过与RPA的多个结构域相互作用而发挥调节作用 并在构象上抑制了该复合体。这起到了抑制RPA-RIP相互作用的作用。vbl.使用 复杂的生物物理、生化和结构工具我证明了单链DNA与RPA-Rtt105复合体结合 并取消限制，促进复员方案因素的招募。在高等真核生物中，一种名为RPAIN的蛋白质 (RPA相互作用蛋白)作为Rtt105的功能同源基因，我将专注于破译其 作用机制。此外，使用低温电子显微镜，我将确定与这些伴侣结合的RPA的结构- 像蛋白质一样。在提案的K00部分，我将重点识别其他 与癌症相关的DDR蛋白，如BRCA1、BRCA2、RAD52和PALB2。此外，我将调查 控制DDR因子在DNA过程中核质分布的调控和信号机制 损坏。最后，利用从生化和细胞研究中获得的知识，我将制定有针对性的 调节DDR的小分子肿瘤治疗抑制剂。F99和K00联合培训阶段将 为我提供必要的技能，使我能够从事独立的研究工作，专注于产生有针对性的 癌症治疗学。

SUMMARY Lesions, breaks, and errors in DNA are drivers of genomic instability resulting in a variety of cancers. The DNA damage response (DDR) is a signaling cascade that responds to breaks in the DNA and utilizes an array of DNA repair factors to correct the error and preserve genomic integrity. Cellular responses to DDR involve regulation of the cell cycle and signaling processes that either trigger DNA repair or programmed cell death. Proteins that function in DDR are shuttled into the nucleus in response to DNA damage. My PhD thesis work and the proposed goals center around understanding the mechanisms that regulate nuclear-cytoplasmic localization of DDR proteins. The F99 part of the proposal focuses on the regulation of Replication Protein A (RPA) by chaperone-like proteins. RPA is an essential single-stranded DNA (ssDNA) binding DDR factor that regulates all aspects of DNA metabolism including DNA replication, repair, and recombination. RPA is transported into the nucleus, recognizes, and binds ssDNA, and activates DDR by interacting with over three dozen RPA-interacting proteins (RIPs). How spurious RPA-RIP interactions are prevented in the cell in the absence of ssDNA has been a long-standing mystery. I have uncovered that Rtt105 (Regulator of Ty1 transposition 105), a chaperone-like protein, functions as a regulator by interacting with multiple domains of RPA and conformationally restraining the complex. This serves as an inhibitor of RPA-RIP interactions. Using sophisticated biophysical, biochemical, and structural tools I show that ssDNA binds to the RPA-Rtt105 complex and removes the restraints to promote recruitment of DDR factors. In higher eukaryotes, a protein called RPAIN (RPA-interacting protein) serves as the functional ortholog of Rtt105 and I will focus on deciphering its mechanism of action. In addition, using cryoEM, I will determine the structures of RPA bound to these chaperone- like proteins. In the K00 part of the proposal, I will focus on identifying chaperone-like proteins specific to other cancer-related DDR proteins such as BRCA1, BRCA2, RAD52, and PALB2. In addition, I will investigate the regulatory and signaling mechanisms that control nuclear-cytoplasmic distribution of DDR factors during DNA damage. Finally, using knowledge obtained from the biochemical and cellular studies I will develop targeted small molecule cancer therapeutic inhibitors to regulate DDR. The combined F99 and K00 training phases will provide me with the necessary skills towards an independent research career focused on generating targeted cancer therapeutics.