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

该项目侧重于氧化DNA碱基修饰7,8-二氢-8-氧鸟嘌呤（8-oxoG）对基因表达的影响，以及在不同序列背景下，在多个损伤位点（聚集性病变）和功能不同的基因元件中8-oxoG的修复特性。在项目的先前阶段，我们已经研究了转录DNA中单个和集群8-oxoG的细胞加工。为此，我们设计了适合于序列特异性结合合成DNA链的报告载体，这些DNA链在特定位置含有8-oxoG病变。通过对小鼠和人类细胞的定量表达分析，我们发现碱基切除修复（BER）反应会影响基因转录，而未切除的8-oxoG耐受性良好。通过化学和遗传手段对早期解核BER活性的操纵，我们进一步发现8-氧鸟嘌呤DNA糖基化酶（OGG1）和无尿嘧啶/无嘧啶核酸内切酶（APE1）都是抑制转录所必需的。引人注目的是，我们发现人类OGG1的8-oxoG碱基切除（整个BER反应的第一步和限速步骤）受到损伤聚类和局部DNA序列背景的强烈影响。另一方面，距离转录起始点不同距离的8-oxoG或位于不同DNA链上的8-oxoG的切除率相当相似。进一步的结果表明，流产的BER反应中间体的加工可能被引导到另一种修复途径中，可能是核苷酸切除修复（NER）。为了继续这项工作，我们计划在转录调控元件中生成含有8-oxoG的构建体，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的相关性，特别是NER的亚途径，用于修复转录基因区域和调控启动子元件中停滞的8-oxoG的BER中间体。为此，我们将使用抗BER（由5'-硫代酸键保护）合成的8-oxodG和ap位点类似物作为模型BER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.