Einfluss von Position und lokaler Häufung (Clustering) der oxidativen DNA-Basenmodifikation 7,8-Dihydro-8-oxoguanin auf Genexpression und DNA-Reparatur in Säugerzellen
Public Health, Gesundheitsbezogene Versorgungsforschung, Sozial- und Arbeitsmedizin
Zusammenfassung der Projektergebnisse
During the third funding period, we explored functions of a major DNA oxidation product and base excision repair (BER) substrate 8-oxo-7,8-dihydroguanine (8-oxoG) at common gene regulatory elements and explored potential connections between BER and other repair pathways. We constructed gene reporters under the control of minimal promoters, whose upstream regulatory regions were replaced by a single binding site for transcriptional activators (CREB, Sp1, or AP-1) and modified to allow incorporation of synthetic 8-oxoG at specific positions. To of the investigated regulatory elements CRE (bound by CREB) and GC box (bound by Sp1) produced sufficient activation over the basal expression level, allowing to measure effects of the incorporated 8-oxoG. Two modes of action were observed in both promoter types. In the absence of BER, 8-oxoG induced mild decrease of promoter activities, attributable to reduced binding affinities of transcription factors to their target motifs. Under conditions when DNA glycosylase OGG1 was depleted, effects of 8-oxoG were steady at all positions analysed and persisted over prolonged time intervals, suggesting that repair was essentially absent. In contrast, in the presence of physiological OGG1 levels, excision of 8- oxoG was followed by a dynamic decrease of promoter activity, which required not only the base excision but also subsequent strand cleavage by APE1. This role of APE1 was demonstrated when 8-oxoG was substituted for a synthetic apurinic site analogues in the presence of either cleavable (phosphodiester) or APE1-resistant (phosphorothioate) 5′-linkage. Both interference with recruitment of transcriptional activators and transcriptional repression promoted by strand cleavage resemble responses to pyrimidine modifications 5-formylcytosine and 5-carboxycytosine, which arise by regulated enzymatic oxidation within the active DNA demethylation pathway (analysed in a parallel project). Remarkably, at two of the tested positions in GC box (both flanked by G on the 3′), 8-oxoG was refractory to excision by OGG1 and, accordingly, did not promote promoter silencing as at other positions. The observed context-specific stability of 8-oxoG indirectly supports recent hypothesis about its plausible epigenetic function, first proposed by Burrows and-co-workers. Investigation of concurrent repair activities towards 8-oxoG, in addition to BER, in transcribed DNA by a host cell reactivation (HCR) approach did not provide a conclusive result because of a low magnitude of the observed effects. However, APE1-resistent AP lesion (tetrahydrofuran protected by uncleavable phosphorothioate linkage) displayed significant transcription blockage if placed in transcribed strand of the reporter gene and its HCR was clearly enhanced by NER. This encouraged us to develop an improved detection principle based on transcriptional miscoding properties (TM) of DNA modifications. We have first established TM assay for AP lesion as a model for a post-excision BER product. The obtained results not only corroborated the evidence of NER, but also provided quantitative estimates for relative efficiencies of the global genome versus transcription-coupled NER of AP sites. Finally, we adapted TM assay to sensitive detection of 8-oxoG, paving the way for and obtained results indicating that clearance of miscoding 8-oxoG is significantly impaired in the presence of phosphorothioate. We also observed enhanced TM in a NER deficient XP-A cell line, although this result has yet to be independently validated in an isogenic cell model with an equivalent OGG1 activity.
Projektbezogene Publikationen (Auswahl)
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Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter. Nucleic Acids Research, 45(19), 11033-11042.
Kitsera, Nataliya; Allgayer, Julia; Parsa, Edris; Geier, Nadine; Rossa, Martin; Carell, Thomas & Khobta, Andriy
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Nucleotide excision repair of abasic DNA lesions. Nucleic Acids Research, 47(16), 8537-8547.
Kitsera, Nataliya; Rodriguez-Alvarez, Marta; Emmert, Steffen; Carell, Thomas & Khobta, Andriy
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EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions. Biomolecules, 10(6), 902.
Rodriguez-Alvarez, Marta; Kim, Daria & Khobta, Andriy
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Regulation of GC box activity by 8-oxoguanine. Redox Biology, 43, 101997.
Müller, Nadine & Khobta, Andriy
