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，并受到来自细胞内外的持续和有害的损害。幸运的是，精心设计的DNA修复系统，包括多功能的核苷酸切除修复（NER），已经发展到克服基因组不稳定性的后果-人类癌症的一个已知标志。然而，基因组修复是复杂的，因为DNA损伤必须迅速进入细胞核的高度浓缩的染色质内。因此，研究建议扩大我们正在进行的工作的范围，通过纳入DNA修复和相关领域的最新发展，特别是染色质重塑的NER的调节。该项目的前提是细胞依赖于专门的分子装置来修改/重塑/重排染色质结构，以克服抑制性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。所涉及的具体假设是：（i）在损伤诱导之后，例如，通过紫外线（UV）照射，核小体在未损伤和损伤的染色质位点上差异重排，（ii）两种主要UV光损伤，环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮光产物（6-4PP）的慢和快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，和（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。拟议的工作将利用相关的大量最先进的技术来解决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中NER的过程，（2）辨别UV照射后未受损和受损染色质中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在CPD和6-4PP修复中的参与，（4）描绘UPS相关染色质重塑在不同UV光损伤的NER中的作用，以及（5）理解检查点信号传导的相互作用，UV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，这些细胞过程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.