Effect of position and clustering of the oxidative DNA base lesion 7,8-dihydro-8-oxoguanine on gene expression and repair in mammalian cells

氧化DNA碱基损伤7,8-二氢-8-氧代鸟嘌呤的位置和聚类对哺乳动物细胞基因表达和修复的影响

• 批准号: 157391409
• 负责人: Professor Dr. Andriy Khobta
• 金额: --
• 依托单位: Institut für Ernährungswissenschaften
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/157391409?language=en
• 关键词: Effect; position; clustering; oxidative; DNA

The project focuses on the effects of the oxidative DNA base modification 7,8-dihydro-8-oxoguanine (8-oxoG) on gene expression and on the peculiarities of repair of 8-oxoG in different sequence contexts, within multiple damage sites (clustered lesions) and within functionally different gene elements. At the previous stages of the project, we have investigated cellular processing of single and clustered 8-oxoG positioned in the transcribed DNA. For this purpose, we have designed reporter vectors suited for sequence specific incorporation of synthetic DNA strands that contained 8-oxoG lesions in defined positions. By quantitative expression analyses in mouse and human cells, we found that base excision repair (BER) reactions affect gene transcription, whereas unexcised 8-oxoG is well tolerated. By manipulation of early nucleolytic BER activities by chemical and genetic means, we further showed that both the 8-oxoguanine DNA glycosylase (OGG1) and the apurinic/apyrimidinic endonuclease (APE1) are required for the inhibition of transcription. Strikingly, we found that 8-oxoG base excision by human OGG1 (the first and rate limiting step of the whole BER reaction) is strongly influenced by damage clustering and local DNA sequence context. On the other hand, excision rates are rather similar for 8-oxoG situated at various distances from the transcription start or in different DNA strands. Further results suggested that processing of abortive BER reaction intermediates may be channeled into an alternative repair pathway, plausibly, nucleotide excision repair (NER). In continuation of this work, we are planning to generate constructs containing 8-oxoG within the transcriptional regulatory elements and investigate the functional impact of 8-oxoG and its repair on gene transcription driven by several families of transcription factors (including CREB, Sp1/Sp2 and Jun). Moreover, we will examine the relevance of alternative repair pathways, in particular subpathways of NER, for the repair of stalled BER intermediates of 8-oxoG in the transcribed gene region and in the regulatory promoter elements. To this purpose, we will deploy BER-resistant (protected by 5'-phosphorothioate linkages) synthetic 8-oxodG and AP-site analogs as model BER intermediates.

该项目的重点是氧化DNA碱基修饰7，8-二氢-8-氧代鸟嘌呤（8-oxoG）对基因表达的影响，以及在不同序列背景下，在多个损伤位点（簇状病变）和功能不同的基因元件内修复8-oxoG的特性。在该项目的前几个阶段，我们研究了位于转录DNA中的单个和簇状8-oxoG的细胞加工。为此，我们设计了适合于序列特异性掺入合成DNA链的报告载体，所述合成DNA链在限定位置含有8-oxoG损伤。通过在小鼠和人类细胞中的定量表达分析，我们发现碱基切除修复（BER）反应影响基因转录，而未切除的8-oxoG耐受性良好。通过化学和遗传手段对早期溶核BER活性的操纵，我们进一步表明，8-氧代鸟嘌呤DNA糖基化酶（OGG 1）和脱嘌呤/脱嘧啶核酸内切酶（APE 1）都是抑制转录所必需的。引人注目的是，我们发现，8-oxoG碱基切除人OGG 1（整个BER反应的第一步和限速步骤）的强烈影响，损害聚类和当地的DNA序列上下文。另一方面，切除率是相当相似的8-oxoG位于不同的距离从转录开始或在不同的DNA链。进一步的结果表明，失败的BER反应中间产物的加工可能被引导到另一个修复途径，可能是核苷酸切除修复（NER）。在这项工作的继续中，我们计划在转录调控元件内产生含有8-oxoG的构建体，并研究8-oxoG及其修复对由几个转录因子家族（包括CREB，Sp1/Sp2和Jun）驱动的基因转录的功能影响。此外，我们将研究替代修复途径的相关性，特别是NER的子途径，用于修复转录基因区域和调控启动子元件中的8-oxoG停滞BER中间体。为此目的，我们将部署BER抗性（由5 '-硫代磷酸酯键保护）合成的8-oxodG和AP位点类似物作为模型BER中间体。