DNA Repair in a Hormone Responsive Gene

描述（由申请人提供）：本提案的主要目的是了解染色质中DNA包装和基因转录背景下DNA修复的分子细节。修复蛋白代表了基因组完整性的“分子监护人”，似乎是某些人类疾病（包括自发性和环境诱导的癌症）的第一道防线之一。DNA损伤是由各种各样的环境因素造成的，如紫外线辐射和化学致癌物，引起基因序列的遗传变化。紫外线辐射和硫酸二甲酯将分别作为原型环境因子用于核苷酸切除修复（NER）和碱基切除修复（BER）的研究。我们将研究DNA修复，转录和染色质结构的不同类别的基因在明确的蛋白质复合物在体外，以及在完整的酵母细胞之间的关系。将在小鼠病毒基因启动子（LTR）的一部分中检查修复，该启动子包装在定位的核小体中，并在体内结合糖皮质激素受体（HR）后诱导。已经合成了DNA中UV损伤的主要形式（CTD），并将其掺入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包装到核小体中，用于研究核小体中CTD位置和方向对非洲爪蟾提取物中NER效率的影响。G：：U错配也将被合成到LTR序列中，以使用纯化的人BER蛋白和哺乳动物细胞提取物来检查HR结合蛋白、q和核小体形成对BER效率的影响。还将在充分表征的诱导型酵母RNA pol II基因（Gal 1 -10和PH 05）以及活性和非活性核糖体RNA pol I基因（rDNA）中检查UV损伤（CPD）的NER和N-甲基嘌呤（NMP）的BER。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细地绘制出来。我们将检查组蛋白基因突变体（sin和Irs）中CPD的全局基因组修复（GGR）和转录偶联修复（TCR）的效率，以及NMP的BER，这些突变体在基因激活期间显示出较少的染色质重塑需要，并且其核小体在体外DNA上更具“移动的”。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中更有效地起作用，在完整细胞中提供DNA修复和染色质重塑之间的联系。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性表型所需的特定基因的表达，这些研究应提供深入了解细胞的防御机制，以抵抗环境致癌物的转化。

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.