DNA repair pathway coordination during damage processing

损伤处理过程中 DNA 修复途径的协调

• 批准号: 10748479
• 负责人: Matthew Allen Schaich
• 金额: $ 7.18万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748479
• 关键词: Address; Aging; Air Pollution; Base Excision Repairs; Binding; Biochemical; CHD1 gene; Carcinogen exposure; Cell Death; Cells; Cellular biology; Chromatin; Complement; DNA; DNA Binding; DNA Damage; DNA Repair Pathway; DNA Sequence Alteration; DNA glycosylase; DNA-Binding Proteins; Disease; Environment; Environmental Exposure; Event; Excision Repair; Exhibits; Exposure to; Fellowship; Frequencies; Human; Individual; Kinetics; Label; Length; Lesion; Malignant Neoplasms; Measures; Neurodegenerative Disorders; Nucleosomes; Nucleotide Excision Repair; Outcome; Pathway interactions; Pollution; Poly(ADP-ribose) Polymerases; Positioning Attribute; Process; Proteins; RAD23B gene; Reactive Oxygen Species; Site; Slide; Smoking; Source; Specificity; System; Testing; Therapeutic Intervention; Time; Toxic Environmental Substances; UV induced DNA damage; Ultraviolet Rays; Water Pollution; Work; Xeroderma Pigmentosum; base; carcinogenicity; cell motility; citrate carrier; cullin 4A; environmental agent; experimental study; fluorescence microscope; improved; knock-down; laser tweezer; metaplastic cell transformation; novel; optic trap; optic tweezer; optical traps; oxidative damage; prevent; repaired; scaffold; sensor; single molecule; therapeutic development; ultraviolet damage; vaping

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.

项目总结/摘要 紫外线、空气污染和其他环境因素中的致癌物质暴露会损害DNA。 由此产生的碱基损伤，如果没有得到适当的处理和去除，可能导致DNA突变， 导致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。虽然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， 最近的研究表明，关键的损伤传感器蛋白在一次修复中并不是孤立的， 途径，但在BER和NER中协同工作：这些包括聚（ADP-核糖）聚合酶1 （PARP 1）（10-17）、UV损伤的DNA结合蛋白（UV-DDB; DDB 1/DDB 2的异二聚体）（18- 18） 20）和着色性干皮病互补C组蛋白（XPC-RAD 23 B）。我假设 关键的损伤传感器蛋白在多种形式的损伤上共同作用，以促进BER， NER，切换机制取决于DNA损伤的类型，并且切换对于 核小体内的加工损伤。这一假设将用单分子进行检验 和细胞生物学实验，完成以下目标：目的1：确定如何 DNA损伤传感器在UV光产物中合作。在子目标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UV光产物上的UV-DDB、XPC-RAD 23 B和PARP 1。Subaim 1b将进行调查 通过成对研究这三种蛋白质的损伤传递动力学（在相同的时间点上）， 浓度）。在子目标1c中，这三种蛋白质的结合参数 将在具有受损DNA的核小体上进行评估，单独分析蛋白质或 相关的核小体相互作用蛋白（HPF 1用于PARP 1，Cullin-4A/RBX用于UV-DDB）。目标2： 定义氧化损伤中DNA损伤的处理和传递，包括单链 断裂和8-oxoG。Subaim 2a将研究PARP 1、XPC-RAD 23 B和UV-DDB在 8-oxoG和单链断裂在单分子水平。每种蛋白质都将被检测 单独地和成对地用于每个受损的衬底。Subaim 2b将测量结合动力学 对于具有8-oxoG和位于三个不同位置的单链断裂的核小体， 网站. Subaim 2c将研究特异性引入活细胞染色质化DNA的8-oxoG 利用新的化学光遗传学系统（18，20，27）。染色质重塑与 破坏部位的解压缩将被击倒，修复动力学将被研究， 损伤传感蛋白、8-氧代鸟嘌呤糖基化酶和其它下游修复蛋白。