Regulation of DNA Excision Repair in Chromatin

染色质 DNA 切除修复的调控

• 批准号: 10227004
• 负责人: John J Wyrick
• 金额: $ 34.33万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227004
• 关键词: Address; Affect; Aging; Alkylation; Award; Base Excision Repairs; Cancer Etiology; Cells; Chromatin; Chromatin Fiber; Chromatin Remodeling Factor; Cisplatin; Complex; Coupled; DNA; DNA Damage; DNA Packaging; DNA Repair; DNA lesion; Data; Enzymes; Eukaryota; Eukaryotic Cell; Excision Repair; Foundations; Genetic Transcription; Genome; Genomics; HTATIP gene; Higher Order Chromatin Structure; Histone Acetylation; Histones; Human; Human Genome; In Vitro; Investigation; Laboratories; Lesion; Life; Location; Maintenance; Malignant Neoplasms; Maps; Measures; Methods; Methyl Methanesulfonate; Modeling; Molecular; Mutation; National Institute of Environmental Health Sciences; Nucleosomes; Nucleotide Excision Repair; Nucleotides; Pathway interactions; Play; Post-Translational Protein Processing; Prevention; Publishing; Pyrimidine Dimers; Reactive Oxygen Species; Regulation; Resolution; Role; Site; Source; System; Testing; UV induced; Ultraviolet Rays; Uracil; Variant; Yeasts; base; chromatin remodeling; cytotoxic; density; design; enzyme activity; genome integrity; genome-wide; histone acetyltransferase; histone modification; insight; methylpurine; non-histone protein; novel; prevent; recruit; repair enzyme; repaired; response; tumor progression; ultraviolet; ultraviolet damage; ultraviolet lesions; yeast genome

PROJECT SUMMARY Maintenance of genomic integrity is fundamental to life. DNA damage occurs spontaneously and ubiquitously from endogenous (e.g., reactive oxygen species) and environmental sources (e.g., ultraviolet (UV) light), inflicting mutagenic and cytotoxic lesions upon the genome that drive the progression of cancer and aging. Cellular excision repair (ER) pathways, including base excision repair (BER) and nucleotide excision repair (NER), are a critical 'first line of defense' responsible for recognizing and removing DNA lesions. The overall objective of this proposal is to understand how ER pathways access DNA lesions that are 'buried' in different types of genomic chromatin. Previous studies have shown that histone post-translational modifications (PTMs) and ATP-dependent chromatin remodelers (ACR) are important for ER of DNA lesions in chromatin. However, it is not known how these chromatin remodeling activities and histone PTMs operate during repair on the diverse spectrum of distinct chromatin types in eukaryotic cells. To address this question, we have developed genome-wide methods to map the formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) and methyl methanesulfonate (MMS)-induced N-methylpurine (NMP) base lesions. Our published study and preliminary data indicate that the CPD-seq and NMP-seq methods can be used to map DNA lesions across the yeast and human genomes at single nucleotide resolution. To better understand the genomic roles of ACRs in ER, we will use the CPD-seq and NMP-seq methods to measure repair in yeast and human cells depleted of different classes of ACRs (Aim I). Histone acetylation is an important PTM associated with DNA damage responses. Our preliminary data suggest that the Esa1/TIP60 histone acetyltransferase (HAT) complex plays a novel role in ER. To test this hypothesis, we will characterize the roles of Esa1/TIP60 and other HATs in regulating ER in both yeast and human cells (Aim II). Our preliminary data indicate that histone acetylation activity in cell-free repair extracts is important for repair of base lesions occluded in nucleosomes. These findings provide the foundation of Aim III, which will identify histone PTMs associated with BER, and characterize their functional role in BER of nucleosomes. Finally, we will investigate the detailed molecular mechanisms by which histone acetylation and ACRs regulate the activity of purified BER enzymes on mononucleosome and oligonucleosome substrates in vitro containing 'designed' DNA base lesions (Aim IV). This proposal is an ongoing investigation of the effects of DNA packaging in chromatin on the two major ER pathways (NER and BER) found in cells. As all eukaryotes, including humans, must deal with this `packaging paradox' for surveillance of the genome, results from these studies are relevant to the broad spectrum of cancer etiology, prevention and treatment.

项目摘要 基因组完整性的维持是生命的基础。 DNA损伤自发和普遍存在 来自内源性（例如，活性氧）和环境源（例如紫外线（UV）光）， 在驱动癌症和衰老进展的基因组上造成诱变和细胞毒性病变。 细胞切除修复（ER）途径，包括基础切除修复（BER）和核苷酸切除修复 （ner）是负责识别和去除DNA病变的关键“第一道防线”。 该提案的总体目的是了解ER途径如何访问DNA病变 在不同类型的基因组染色质中“埋葬”。先前的研究表明，翻译后组蛋白 修饰（PTM）和ATP依赖性染色质重塑剂（ACR）对于DNA病变的ER很重要 染色质。但是，尚不知道这些染色质重塑活性和组蛋白PTM如何运作 在真核细胞中不同染色质类型的各种谱系上修复过程中。为了解决这个问题， 我们已经开发了全基因组方法来绘制紫外线诱导的环丁烷的形成和修复 嘧啶二聚体（CPD）和甲基甲磺酸甲酯（MMS）诱导的N-甲基嘌呤（NMP）碱病变。 我们发表的研究和初步数据表明，CPD-SEQ和NMP-SEQ方法可用于 在单核苷酸分辨率下，横跨酵母的DNA病变和人类基因组。更好地理解 ACR在ER中的基因组作用，我们将使用CPD-Seq和NMP-Seq方法来测量酵母的修复 人类细胞耗尽了不同类别的ACRS（AIM I）。组蛋白乙酰化是重要的PTM 与DNA损伤反应有关。我们的初步数据表明ESA1/TIP60组蛋白 乙酰基转移酶（HAT）复合物在ER中起新颖的作用。为了检验这一假设，我们将表征 ESA1/TIP60和其他帽子在调节酵母和人类细胞中ER中的作用（AIM II）。我们的初步 数据表明，无细胞修复提取物中的组蛋白乙酰化活性对于修复碱病变很重要 在核小体中闭塞。这些发现为AIM III提供了基础，该基础将识别组蛋白PTM 与BER相关，并表征其在核小体BER中的功能作用。最后，我们将调查 组蛋白乙酰化和ACR调节纯化的活性的详细分子机制 单核体和寡核小体底物的BER酶体外含有“设计” DNA碱基 病变（AIM IV）。 该建议是对DNA包装在染色质中对两个主要的影响的持续研究 ER途径（NER和BER）在细胞中发现。正如所有真核生物（包括人类）必须处理的那样 “包装悖论”以监视基因组，这些研究的结果与广泛的 癌症病因，预防和治疗的频谱。