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

该项目的重点是氧化DNA碱基修饰7，8-二氢-8-氧鸟嘌呤(8-oxoG)对基因表达的影响，以及8-oxoG在不同序列背景、多个损伤部位(聚集性损伤)和功能不同的基因元件中修复的特点。在项目的前几个阶段，我们已经研究了位于转录DNA中的单个和簇状8-oxoG的细胞处理。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，这些合成DNA链在定义的位置包含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具有良好的耐受性。通过化学和遗传手段控制早期的核溶解BER活性，我们进一步证明8-氧鸟嘌呤DNA糖基酶(OGG1)和脱嘌呤/脱嘧啶核酸内切酶(APE1)都是抑制转录所必需的。值得注意的是，我们发现人OGG1(整个BER反应的第一步也是限速步骤)对8-oxoG碱基的切除受到损伤聚集和局部DNA序列背景的强烈影响。另一方面，位于转录起始位置不同距离或位于不同DNA链中的8-oxoG的切除率非常相似。进一步的结果表明，对流产的BER反应中间产物的处理可能被引导到另一条修复途径，似乎是核苷酸切除修复(NER)。在继续这项工作的基础上，我们计划构建包含8-oxoG的转录调控元件，并研究8-oxoG及其修复对几个转录因子家族(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，特别是NER亚途径，与转录基因区域和调控启动子元件中8-oxoG停滞的BER中间产物修复的相关性。为此，我们将部署抗误码率(受5‘-硫代硫酸键保护)的合成8-oxodG和AP位类似物作为误码率模型中间体。

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.