Repair of Genotoxic Damage in Chromatin

描述（由申请人提供）：生物体的基因组本质上是不稳定的，并且受到来自细胞内外的持续和有害的损害。幸运的是，复杂的DNA修复系统，包括多功能核苷酸切除修复（NER），已经进化到能够克服基因组不稳定的后果——这是人类癌症的已知标志。然而，基因组修复是复杂的，因为DNA损伤必须在细胞核高度浓缩的染色质内迅速进入。因此，我们建议通过结合DNA修复和相关领域的最新进展，特别是染色质重塑，来扩展我们正在进行的关于NER调控的研究范围。该项目的前提是，细胞依靠专门的分子装置来修饰/重塑/重排染色质结构，以克服抑制染色质结构所带来的障碍，从而允许所有DNA模板化过程的操作，包括DNA修复。具体的假设是：(i)损伤诱导后，如紫外线（UV）照射，核小体在未损伤和受损染色质的位置上重排不同；（ii）两种主要的紫外线光损伤，环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶光产物（6-4PP）的缓慢和快速修复速率依赖于不同的染色质重塑机制；（iii）泛素-蛋白酶体途径（UPS）密切调节NER期间的染色质重塑。（iv）切除修复结果由DNA损伤反应和染色质重塑之间的相互作用决定。拟议的工作将利用大量相关的最新技术来实现以下相互关联的具体目标：(1)证明组蛋白修饰参与哺乳动物细胞NER过程；(2)识别紫外线照射后未损伤和受损染色质的核小体重排及其对NER因子结合的影响；(3)显示染色质重塑机制参与CPD和6-4PP的修复；(4)描述UPS相关染色质重塑在不同紫外线光照射下NER中的作用；(5)了解检查点信号的相互作用。紫外线照射后染色质重塑和NER因子募集。这些研究将为复杂的细胞过程提供开创性的见解，以保持基因组的完整性并防止人类癌症的发生。

DESCRIPTION (provided by applicant): Genome of living organisms is intrinsically unstable and subjected to incessant and deleterious damage from within and outside of cells. Fortunately, elaborate DNA repair systems, including the versatile nucleotide excision repair (NER) have evolved to overcome the consequences of genomic instability- a known hallmark of human cancer. Nevertheless, genomic repair is complicated because the DNA lesions must be promptly accessed within the highly condensed chromatin of cell's nucleus. Thus, studies are proposed to extend the scope of our ongoing work on the regulation of NER by incorporating the latest developments in DNA repair and related areas, especially chromatin remodeling. The project is based on the premise that cells rely on specialized molecular apparatus to modify/remodel/rearrange chromatin structure to overcome the barrier presented by repressive chromatin structure and thus allowing the operation of all the DNA templated processes, including DNA repair. The specific hypotheses addressed are: (i) subsequent to damage induction, e.g., by ultraviolet (UV) irradiation, nucleosomes are differentially rearranged at the sites of non-damaged and damaged chromatin, (ii) the slow and fast repair rates of two main UV photolesions, cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts (6-4PP), are dependent on different chromatin remodeling machineries, (iii) ubiquitin-proteasome pathway (UPS) intimately regulates chromatin remodeling during NER, and (iv) the excision repair outcome is determined by the interplay between DNA damage response and chromatin remodeling. The proposed work will utilize a relevant plethora of state-of-the art technologies to address following inter-related specific objectives: (1) to demonstrate the involvement of histone modifications in the process of NER in mammalian cells, (2) to discern the nucleosome rearrangement in undamaged and damaged chromatin following UV irradiation and its effect on NER factor binding, (3) to show the participation of chromatin remodeling machinery in the repair of CPD and 6-4PP, (4) to delineate the role of UPS related chromatin remodeling in NER of different UV photolesions, and (5) to understand the interplay of checkpoint signaling, chromatin remodeling and NER factor recruitment following UV irradiation. These studies will provide seminal insights into intricate cellular processes that preserve genomic integrity and prevent initiation of human cancers.