DNA Repair In A Hormone Responsive Gene

描述(由申请人提供)：本提案的广泛目标是在DNA包装和染色质基因转录的背景下了解DNA切除修复的分子细节。我们将分别使用紫外线辐射和DNA甲基化化学品作为原型环境试剂来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)的一部分中进行检测，该基因启动子包装在一个定位的核小体(NCP)中，并在体内通过糖皮质激素受体(HR)结合而诱导。在目标I中，我们将研究NCP解缠动力学在推动修复蛋白和DNA损伤之间的复杂形成中的作用。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解缠动力学的影响。DNA中紫外线损伤的主要形式(CTD)和G：：U错配将被结合在含有GRE的NCP的特定位置，并通过将GRE序列与NCP定位元件括在一起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装NCPs的速率。我们还将检测在GRE与蛋白质复合体(AIM II)中特定位置结合的尿嘧啶的误码率。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位置、组蛋白修饰和HR结合对GRE和相邻序列中特定位置的尿嘧啶碱基BER的影响。含有G：：U错配的带括号的GRE序列将被包装成含有未修饰或特定修饰的组蛋白的核小体，以便与裸DNA进行比较。紫外线损伤(CPDS)的NER和N-甲基嘌呤(NMPs)的误码率也将在酵母中描述良好的染色质基因座中进行检测。我们将检测组蛋白突变体(SIN和IRS)中CPD的NER的效率，这些突变体在基因激活过程中需要较少的染色质重塑，并且其NCP更具“移动性”(Aim III)。我们将集中于从修复熟练(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)的每条链中去除CPD。这些基因座的染色质结构是众所周知的，它们提供了不同的染色质‘景观’，用于与wt细胞进行比较。最后，我们将检验NMPs在酵母SIN和LRS突变体中的误码率效率(目标IV)。将在wt和rad突变细胞中比较从sin和irs突变体的活性和非活性Pol I和Pol II基因的每条链中去除NMP。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，最终目的是了解人类细胞的这一过程。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。 与公共卫生相关：DNA损伤对所有活细胞都是有害的，修复哺乳动物细胞中的这些损伤是对抗突变和癌症的“一线防御”。我们使用原型DNA损伤剂来诱导细胞中DNA包装的不同区域的DNA修复，以确定细胞如何应对DNA中“埋藏”的损伤的修复。因此，我们实验室的结果对癌症的病因、预防和治疗具有广泛的意义。

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. PUBLIC HEALTH RELEVANCE: DNA damage is detrimental to all living cells and repair of these lesions in mammalian cells is a 'frontline defense' against mutations and cancer. We use prototype DNA-damaging agents to elicit DNA repair in different regions of DNA packaging in cells to determine how cells cope with repair of 'buried' lesions in DNA. Thus, results from our laboratory have implications for the broad spectrum of cancer etiology, prevention and treatment.