Regulation of DNA Excision Repair in Chromatin

项目总结 保持基因组的完整性是生命的根本。DNA损伤是自发且无处不在的 来自内源(例如，活性氧种)和环境源(例如，紫外线(UV)光)， 对基因组造成突变和细胞毒性损伤，从而推动癌症和衰老的进展。 细胞切除修复途径，包括碱基切除修复和核苷酸切除修复 (NER)是负责识别和移除DNA损伤的关键“第一道防线”。 该提案的总体目标是了解ER通路如何访问DNA损伤 “埋藏”在不同类型的基因组染色质中。先前的研究表明，组蛋白翻译后 修饰(PTM)和ATP依赖的染色质重构体(ACR)对DNA损伤的ER是重要的 染色质。然而，目前还不知道这些染色质重塑活动和组蛋白PTM是如何运作的。 在修复期间，真核细胞中不同类型的染色质的不同光谱。为了解决这个问题， 我们已经开发了全基因组的方法来绘制紫外线诱导的环丁烷的形成和修复的图谱 嘧啶二聚体(CPDS)和甲基磺酸甲酯(MMS)诱导的N-甲基嘌呤(NMP)碱基损伤。 我们已发表的研究和初步数据表明，CPD-SEQ和NMP-SEQ方法可以用于 在单核苷酸分辨率下绘制酵母和人类基因组中的DNA损伤图。为了更好地理解 ACRs在内质网中的基因组作用，我们将使用CPD-seq和NMP-seq方法来测量酵母中的修复 和人类细胞耗尽不同类别的ACRs(目标I)。组蛋白乙酰化是一种重要的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)复合体在内质网中发挥着新的作用。为了检验这一假设，我们将描述 ESA1/Tip60和其他HATS在调节酵母和人类细胞内质网中的作用(目标II)。我们的预赛 数据表明，无细胞修复提取物中的组蛋白乙酰化活性对碱基损伤的修复很重要。 闭塞在核小体中。这些发现为AIM III提供了基础，AIM III将识别组蛋白PTM 与误码率相关，并表征它们在核小体误码率中的功能作用。最后，我们将调查 组蛋白乙酰化和ACRs调节纯化蛋白活性的详细分子机制 含有DNA碱基的单核小体和寡核小体底物上的BER酶 损害(目标IV)。 这项提议是对染色质中DNA包装对两个主要 在细胞中发现了ER通路(NER和BER)。因为所有真核生物，包括人类，都必须处理这一问题 “包装悖论”对于基因组的监测，这些研究的结果与广泛的 癌症病因学、预防和治疗的光谱。

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.