DNA Repair in a Hormone Responsive Gene

描述（由申请人提供）：本提案的主要目标是了解DNA包装和染色质基因转录背景下DNA修复的分子细节。修复蛋白代表了基因组完整性的“分子守护者”，似乎是对抗某些人类疾病的第一道防线之一，包括自发性和环境诱发的癌症。DNA损伤是由多种环境因素引起的，如紫外线辐射和化学致癌物，导致基因序列的遗传变化。紫外线辐射和硫酸二甲基将分别作为研究核苷酸切除修复（NER）和碱基切除修复（BER）的原型环境剂。我们将研究DNA修复，转录和染色质结构之间的关系在明确定义的蛋白质复合物的不同类别的基因在体外，以及在完整的酵母细胞。修复将在小鼠病毒基因启动子（LTR）的一个片段中进行检查，该启动子被包装在一个定位的核小体中，并在体内与糖皮质激素受体（HR）结合后诱导。DNA中紫外线损伤的主要形式（CTD）已经被合成，并将被纳入LTR的特定位点，用于研究CTD损伤对HR结合的影响。该序列还将被核小体定位元件包围并打包成核小体，用于研究CTD在核小体中的位置和取向对爪蟾提取物NER效率的影响。G::U错配也将合成到LTR序列中，以检测HR结合蛋白、q和核小体形成对BER效率的影响，使用纯化的人BER蛋白和哺乳动物细胞提取物。我们还将在特征明确的可诱导酵母RNA pol II基因（Gal1-10和PH05）以及活性和非活性核糖体RNA pol I基因（rDNA）中检测紫外线损伤的NER （CPDs）和n -甲基嘌呤的BER （NMPs）。这些不同基因位点的染色质结构在非活性和活性基因状态下都被精确地绘制出来。我们将研究组蛋白基因突变体（sin和Irs）中cpd的全局基因组修复（GGR）和转录偶联修复（TCR）以及nmp的BER的效率，这些突变体在基因激活过程中对染色质重塑的需求较少，其核小体在体外DNA上更具“流动性”。因此，NER （GGR或TCR）和/或BER可能在这些突变体中定位的核小体区域更有效地发挥作用，在完整细胞中提供DNA修复和染色质重塑蛋白之间的联系。因此，我们将研究基因表达和局部染色质结构变化对DNA修复效率的影响。由于这些病变可能会改变建立肿瘤表型所需的特定基因的表达，因此这些研究应有助于深入了解细胞抵抗环境致癌物转化的防御机制。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to understand the molecular details of DNA repair in the context of DNA packaging and gene transcription in chromatin. The repair proteins represent 'molecular guardians' of genomic integrity and appear to be one of the first lines of defense aqainst certain human diseases, including both spontaneous and environmentally induced cancers. DNA damage results from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens, giving rise to heritable changes in the sequence of genes. UV radiation and dimethylsulfate will be used as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. We will examine the relationship between DNA repair, transcription and chromatin structure of different classes of genes in well-defined protein complexes in vitro, as well as in intact yeast cells. Repair will be examined in a section of a mouse viral gene promoter (LTR), that is packaged in a positioned nucleosome and induced upon binding of a glucocorticoid hormone receptor (HR) in vivo. The major form of UV damage in DNA (CTD) has been synthesized and will be incorporated into specific sites of the LTR for studies on the effect of CTD damage on HR binding. This sequence will also be bracketed by nucleosome positioning elements and packaged into a nucleosome for studies on the effect of CTD location and orientation in a nucleosome on NER efficiency in Xenopus extracts. G::U mismatches will also be synthesized into the LTR sequence to examine the effect of HR bindin,q and nucleosome formation on BER efficiency, using purified human BER proteins and mammalian cell extracts. NER of UV damaqe (CPDs) and BER of N-methyl purines (NMPs) will also be examined in well-characterized, inducible yeast RNA pol II genes (Gal1-10 and PH05), and active and inactive ribosomal RNA pol I genes (rDNA). The chromatin structure of these different gene loci has been exquisitely mapped in both the inactive and active gene states. We will examine the efficiency of global genome repair (GGR) and transcription coupled repair (TCR) of CPDs, as well as BER of NMPs, in histone gene mutants (sin and Irs) that show less need for chromatin remodeling during gene activation and whose nucleosomes are more 'mobile' on DNA in vitro. Therefore, NER (GGR or TCR) and/or BER may function more efficiently in regions of positioned nucleosomes in these mutants, providinq a link between DNA repair and chromatin remodelin,q in intact cells. Thus, we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA repair. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phlenotype, these studies should provide insiqht into the cell's defense mechanism for resisting transformation by environmental carcinogens.