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

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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.