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切除修复的分子细节。我们将使用紫外线辐射和DNA甲基化化学物质作为原型环境剂，分别研究核苷酸切除修复（NER）和碱基切除修复（BER）。修复将在小鼠病毒基因启动子（GRE）的一个片段中进行检查，该片段被包装在定位核小体（NCP）中，并在体内被糖皮质激素受体（HR）结合诱导。在目的1中，我们将研究NCP解包裹动力学在驱动修复蛋白和DNA损伤之间复合物形成中的作用。这些研究将涉及使用限制性内切酶可及性（REA）和Fvrster共振能量转移（FRET）来确定DNA损伤对NCP解包裹动力学的影响。DNA中紫外线损伤的主要形式（CTD）和G::U错配将被纳入含有GRE的NCP的特定位点，并通过用NCP定位元件包围GRE序列在组蛋白表面旋转排列。动态FRET将用于监测DNA修复蛋白在特定部位病变处捕获未包裹的ncp的速率。我们还将检查尿嘧啶在GRE中与蛋白质结合的特定位点的BER（目的II）。这些研究将涉及使用纯化的人类酶和哺乳动物细胞提取物来确定核小体定位、组蛋白修饰和HR结合对BER在GRE和邻近序列中特定位点尿嘧啶碱基的影响。括号内含有G::U错配的GRE序列将被打包到含有未修饰或特异性修饰组蛋白的核小体中，以便与裸DNA进行比较。我们还将在酿酒酵母的染色质位点上检测紫外线损伤的内能（CPDs）和n -甲基嘌呤（NMPs）的内能（BER）。我们将研究组蛋白突变体（sin和Irs）中cpd的NER效率，这些突变体在基因激活过程中需要较少的染色质重塑，其ncp更具“流动性”（目的III）。我们将专注于从修复熟练（wt）和修复缺陷（rad）细胞的lrs突变体的活性和非活性Pol II基因和核糖体RNA基因（Pol I）的每条链上去除cpd。这些基因座的染色质结构是众所周知的，它们提供了与wt细胞不同的染色质“景观”。最后，我们将研究NMPs在酵母sin和lrs突变体中的BER效率（aim IV）。将比较wt和rad突变体细胞在sin和Irs突变体中活性和非活性Pol I和Pol II基因各链上nmp的去除情况。因此，我们将使用“多方面”的方法来研究染色质结构在DNA修复中的作用，最终目标是了解人类细胞中的这一过程。由于DNA损伤可能改变建立肿瘤表型所需的特定基因的表达，这些研究也应该为细胞抵抗环境致癌物的肿瘤转化的防御机制提供有价值的见解。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to understand the molecular details of DNA excision repair in the context of DNA packaging and gene transcription in chromatin. We will use UV radiation and DNA methylating chemicals as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. Repair will be examined in a section of a mouse viral gene promoter (GRE) that is packaged in a positioned nucleosome (NCP) and induced upon glucocorticoid hormone receptor (HR) binding in vivo. In aim I, we will examine the role of NCP unwrapping dynamics in driving complex formation between repair proteins and DNA lesions. These studies will involve the use of restriction enzyme accessibility (REA) and Fvrster resonance energy transfer (FRET) to determine the effect of DNA lesions on NCP unwrapping dynamics. The major form of UV damage in DNA (CTD) and G::U mismatches will be incorporated at specific sites of GRE-containing NCPs and rotationally aligned on the histone surface by bracketing the GRE sequence with NCP positioning elements. Dynamic FRET will be used to monitor the rates of trapping of unwrapped NCPs by DNA repair proteins at site-specific lesions. We will also examine BER of uracil incorporated at specific sites in the GRE complexed with protein (aim II). These studies will involve using purified human enzymes and mammalian cell extracts to determine the effect of nucleosome location, histone modification and HR binding on BER at site-specific uracil bases in the GRE and adjacent sequences. The bracketed GRE sequence containing G::U mismatches will be packaged into nucleosomes containing unmodified or specifically modified histones for comparisons with naked DNA. NER of UV damage (CPDs) and BER of N-methyl purines (NMPs) will also be examined in well- characterized chromatin loci in the yeast S. cerevisiae. We will examine the efficiency of NER of CPDs in histone mutants (sin and Irs) that require less chromatin remodeling during gene activation and whose NCPs are more 'mobile' (aim III). We will focus on removal of CPDs from each strand of active and inactive Pol II genes and ribosomal RNA genes (Pol I) of lrs mutants of repair proficient (wt) and repair deficient (rad) cells. The chromatin structure of these loci is well known and they provide different chromatin 'landscapes' for comparison with wt cells. Finally, we will examine the efficiency of BER of NMPs in sin and lrs mutants of yeast (aim IV). Removal of NMPs from each strand of active and inactive Pol I and Pol II genes of sin and Irs mutants, will be compared between wt and rad mutant cells. Thus, we will use a "multifaceted" approach to examine the role of chromatin structure in DNA repair with the ultimate goal of understanding this process in human cells. Since DNA lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should also provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.