DNA repair pathway coordination during damage processing

项目摘要/摘要 紫外线、暴露在空气污染中的致癌物和其他环境因素都会破坏DNA。 由此产生的碱基损伤，如果没有得到适当的处理和去除，可能会导致DNA突变， 引起细胞死亡或细胞转化，表现为多种人类疾病，包括 衰老、癌症和神经退行性疾病。而典型的紫外线损伤和氧化 损伤通过核苷酸切除修复(NER)和碱基切除修复(BER)修复， 最近的研究分别表明，关键的损伤感受器蛋白在一次修复中不是孤立的 途径，但在BER和NER中协同工作：包括聚(ADP-核糖)聚合酶1 (PARP1)(10-17)，紫外线损伤DNA结合蛋白(UV-DDB；DDB1/DDB2的杂二聚体)(18- 20)和色素性干皮病互补C组蛋白(XPC-RAD23B)。我假设 关键的损伤感应器蛋白在多种形式的损伤上协同工作，以促进BER和 此外，移交机制取决于DNA损伤类型，且移交对于 核小体内嵌入的突起损伤。这一假说将用单分子进行检验。 和细胞生物学实验完成了以下目标：目标1：确定如何 DNA损伤传感器在紫外线照相产品中发挥作用。在亚目标1a中，DNA结合位置， 结合寿命和均方位移(对于在DNA上滑动的运动事件)将是 分析了UV-DDB、XPC-RAD23B和PARP1在UV感光产品上的情况。苏巴伊姆1b将进行调查 成对研究这三种蛋白质的损伤转移动力学 浓度)使用正交标签。在亚目的1c中，这三种蛋白的结合参数 将在DNA受损的核小体上进行评估，单独或与蛋白质一起分析 相关的核小体相互作用蛋白(PARP1为HPF1，UV-DDB为cullin-4A/RBX)。目标2： 定义包括单链在内的氧化损伤的DNA损伤处理和移交 休息和8-OXOG。Subaim 2a将研究PARP1、XPC-RAD23B和UV-DDB在 8-oxoG和单链断裂发生在单分子水平。每种蛋白质都会被检测 对于每个损坏的衬底，单独和成对安装。Subaim 2b将测量结合动力学 对于核小体的每种蛋白质，8-oxoG和单链断裂位于三个不同的位置 网站。Subaim 2c将研究专门引入活细胞染色体DNA的8-oxoG 利用新的化学遗传系统(18、20、27)。染色质重构体与 损伤部位的解压将被拆除，修复动力学将被研究 损伤感受器蛋白、8-氧鸟嘌呤糖基酶和其他下游修复蛋白。

Project Summary/Abstract UV light, carcinogen exposure from air pollution and other environmental agents can damage DNA. The resulting base damages, if not properly processed and removed, can lead to DNA mutations, causing cell death or cellular transformation manifested in a wide variety of human maladies including aging, cancer, and neurodegenerative diseases. While canonically UV-damage and oxidative damage are repaired by nucleotide excision repair (NER) and base excision repair (BER), respectively, recent work indicates that key damage sensor proteins are not siloed within one repair pathway but work cooperatively in both BER and NER: these include poly(ADP-ribose) polymerase 1 (PARP1) (10-17), UV-damaged DNA binding protein (UV-DDB; a heterodimer of DDB1/DDB2) (18- 20), and xeroderma pigmentosum complementation group C protein (XPC-RAD23B). I hypothesize that key damage sensor proteins work together on multiple forms of damage to facilitate BER and NER, that handoff mechanisms depend on the type of DNA damage, and that handoff is essential to process damage embedded within nucleosomes. This hypothesis will be tested with single molecule and cell biology experiments with the completion of the following aims: AIM 1: To determine how DNA damage sensors cooperate at UV photoproducts. In subaim 1a, DNA binding positions, binding lifetimes, and mean squared displacement (for motile events that slide on the DNA) will be analyzed for UV-DDB, XPC-RAD23B, and PARP1 on UV photoproducts. Subaim 1b will investigate the kinetics of lesion hand-off for these three proteins by studying them in pairs (at equal concentrations) using orthogonal labels. In subaim 1c, the binding parameters of these three proteins will be assessed on nucleosomes with damaged DNA, with the proteins analyzed alone or with relevant nucleosome interacting proteins (HPF1 for PARP1 and Cullin-4A/RBX for UV-DDB). AIM 2: To define DNA damage processing and handoff at oxidative lesions including single strand breaks and 8-oxoG. Subaim 2a will study the interactions of PARP1, XPC-RAD23B, and UV-DDB on 8-oxoG and single-strand breaks at the single-molecule level. Each protein will be examined individually and in pairs for each damaged substrate. Subaim 2b will measure the binding dynamics for each protein for nucleosomes with 8-oxoG and single-strand breaks positioned at three different sites. Subaim 2c will study 8-oxoG introduced specifically into the chromatinized DNA of living cells utilizing a novel chemoptogenetic system (18, 20, 27). Chromatin remodelers associated with decompaction at damage sites will be knocked down and the repair kinetics will be studied for damage sensor proteins, 8-oxoguanine glycosylase, and other downstream repair proteins.