Regulation of DNA Excision Repair in Chromatin

项目摘要 维持基因组的完整性是生命的基础。DNA损伤是自发的，无处不在 从内源性（例如，活性氧类）和环境源（例如，紫外（UV）光）， 对基因组造成诱变和细胞毒性损伤，从而导致癌症和衰老的进展。 细胞切除修复（ER）途径，包括碱基切除修复（BER）和核苷酸切除修复 (NER)是负责识别和去除DNA损伤的关键“第一道防线”。 这项提案的总体目标是了解ER途径如何进入DNA损伤， “埋”在不同类型的基因组染色质中。以前的研究表明，组蛋白的翻译后 修饰（PTM）和ATP依赖的染色质重塑（ACR）对于ER的DNA损伤是重要的， 染色质然而，尚不清楚这些染色质重塑活动和组蛋白PTM如何运作 在真核细胞中不同染色质类型的多样性谱的修复过程中。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 嘧啶二聚体（CPD）和甲基甲磺酸酯（MMS）诱导的N-甲基嘌呤（NMP）的基础病变。 我们已发表的研究和初步数据表明，CPD-测序和NMP-测序方法可用于 以单核苷酸分辨率绘制酵母和人类基因组的DNA损伤图谱。更好地了解 由于ACR在ER中的基因组作用，我们将使用CPD-seq和NMP-seq方法来测量酵母中的修复 和耗尽不同类别ACR的人细胞（Aim I）。组蛋白乙酰化是一个重要的PTM 与DNA损伤反应有关。我们的初步数据表明Esa 1/TIP 60组蛋白 乙酰基转移酶（HAT）复合物在内质网中起着新的作用。为了验证这一假设，我们将描述 Esa 1/TIP 60和其他HAT在酵母和人类细胞中调节ER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基底损伤的修复是重要的 封闭在核小体中。这些发现为Aim III提供了基础，Aim III将识别组蛋白PTM 与BER相关，并表征它们在核小体BER中的功能作用。最后，我们将调查 组蛋白乙酰化和ACRs调节纯化蛋白活性的详细分子机制 BER酶在含有“设计”DNA碱基的体外单核苷酸和寡核苷酸底物上的作用 病变（Aim IV）。 这项建议是一项正在进行的研究，研究染色质中DNA包装对两个主要的细胞的影响。 ER途径（NER和BER）在细胞中发现。因为所有的真核生物，包括人类， “包装悖论”的基因组监测，从这些研究的结果是相关的广泛 癌症病因学、预防和治疗谱。

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.