Role of nuclear chaperones in genomic instability and carcinogenesis

总结 DNA中的损伤、断裂和错误是导致各种癌症的基因组不稳定性的驱动因素。的 DNA损伤反应（DDR）是一种信号级联反应，其响应DNA中的断裂并利用阵列 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 调节细胞周期和信号传导过程，触发DNA修复或程序性细胞死亡。 在DDR中起作用的蛋白质响应于DNA损伤而穿梭进入细胞核。我的博士论文 提出的目标围绕着理解调节细胞核-细胞质的机制 DDR蛋白的定位。该提案的F99部分侧重于复制蛋白A的调节 (RPA)通过类似伴侣蛋白的蛋白质。RPA是一种必需的单链DNA（ssDNA）结合DDR因子， 调节DNA代谢的所有方面，包括DNA复制、修复和重组。RPA是 转运到细胞核中，识别并结合ssDNA，并通过与三个以上的 12种RPA相互作用蛋白（RIP）。如何在小区中防止虚假RPA-RIP交互 ssDNA的缺失一直是一个长期的谜团。我发现Rtt 105（Ty 1的调节器 转座105），一种伴侣样蛋白，通过与RPA的多个结构域相互作用而起调节剂的作用 并在构象上限制复合物。这是RPA-RIP相互作用的抑制剂。使用 复杂的生物物理，生物化学和结构工具，我表明ssDNA结合RPA-Rtt 105复合物 并消除对促进复员方案人员征聘的限制。在高等真核生物中， （RPA相互作用蛋白）作为Rtt 105的功能直系同源物，我将专注于破译它的功能。 作用机制。此外，利用冷冻电镜，我将确定结合到这些分子伴侣的RPA的结构比如蛋白质。在K 00部分的建议，我将重点确定伴侣蛋白样蛋白的具体到其他 癌症相关DDR蛋白，如BRCA 1、BRCA 2、RAD 52和PALB 2。此外，我将调查 在DNA合成过程中控制DDR因子的核质分布的调节和信号传导机制 损害最后，利用从生物化学和细胞研究中获得的知识，我将开发有针对性的 用于调节DDR的小分子癌症治疗抑制剂。F99和K 00训练阶段将 为我提供了必要的技能，对一个独立的研究生涯，重点是产生有针对性的 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 chaperonelike 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.