Regulation of GC box activity by 8-oxoguanine
The oxidation-induced DNA modification 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) was recently implicated in the activation and repression of gene transcription. We aimed at a systematic characterisation of the impacts of 8-oxodG on the activity of a GC box placed upstream from the RNA polymerase II core promoter. With the help of reporters carrying single synthetic 8-oxodG residues at four conserved G:C base pairs (underlined) within the 5′-TGGGCGGAGC-3′ GC box sequence, we identified two modes of interference of 8-oxodG with the promoter activity. Firstly, 8-oxodG in the purine-rich (but not in the pyrimidine-rich) strand caused direct impairment of transcriptional activation. In addit…
Nucleotide excision repair of abasic DNA lesions
AbstractApurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, w…
Cockayne syndrome: varied requirement of transcription-coupled nucleotide excision repair for the removal of three structurally different adducts from transcribed DNA.
Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER) pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS), however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N 2-yl)-2-acetylaminofluorene adduct (dG(N 2)-AAF) constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is remov…
Excision of Uracil from Transcribed DNA Negatively Affects Gene Expression
Uracil is an unavoidable aberrant base in DNA, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires a succession of intermediate products, including an abasic site and a single strand break, before the original DNA structure can be reconstituted. These repair intermediates are harmful for DNA replication and also interfere with transcription under cell-free conditions. However, their relevance for cellular transcription has not been proved. Here we investigated the influence of uracil incorporated into a reporter vector on gene expression in human cells. The expression constructs contained a single uracil opposi…
Interactions between DNA damage, repair, and transcription
This review addresses a variety of mechanisms by which DNA repair interacts with transcription and vice versa. Blocking of transcriptional elongation is the best studied of these mechanisms. Transcription recovery after damage therefore has often been used as a surrogate marker of DNA repair in cells. However, it has become evident that relationships between DNA damage, repair, and transcription are more complex due to various indirect effects of DNA damage on gene transcription. These include inhibition of transcription by DNA repair intermediates as well as regulation of transcription and of the epigenetic status of the genes by DNA repair-related mechanisms. In addition, since transcript…
Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter
Abstract Enzymatic oxidation of 5-methylcytosine (5-mC) in the CpG dinucleotides to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) has central role in the process of active DNA demethylation and epigenetic reprogramming in mammals. However, it is not known whether the 5-mC oxidation products have autonomous epigenetic or regulatory functions in the genome. We used an artificial upstream promoter constituted of one cAMP response element (CRE) to measure the impact of 5-mC in a hemi-methylated CpG on the promoter activity and further explored the consequences of 5-hmC, 5-fC, and 5-caC in the same system. All modifications induced mild impairment of the …
Repair of oxidatively generated DNA damage in Cockayne syndrome
Defects in the repair of endogenously (especially oxidatively) generated DNA modifications and the resulting genetic instability can potentially explain the clinical symptoms of Cockayne syndrome (CS), a hereditary disease characterized by developmental defects and neurological degeneration. In this review, we describe the evidence for the involvement of CSA and CSB proteins, which are mutated in most of the CS patients, in the repair and processing of DNA damage induced by reactive oxygen species and the implications for the induction of cell death and mutations. Taken together, the data demonstrate that CSA and CSB, in addition to their established role in transcription-coupled nucleotide…
EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions
The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress
8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein
We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantitative real-time PCR to measure the changes in gene expression caused by the presence of oxidised guanine residues generated by photosensitisation in the vector DNA. In wild-type cells, small numbers (one or three) of oxidised guanines did not affect gene expression at short times after transfections, whereas progressive reduction of the transgene expression was observed at later time points. Alth…
Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system
Background Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. Methods To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)).…
Generation of reporter plasmids containing defined base modifications in the DNA strand of choice
Physiological effects of DNA bases other than A, G, C, and T as well as ways of removal of such bases from genomes are studied intensely. Methods for targeted insertion of modified bases into DNA, therefore, are highly demanded in the fields of DNA repair and epigenetics. This article describes efficient procedures for incorporation of modified DNA bases into a plasmid-borne enhanced green fluorescent protein (EGFP) gene. The procedure exploits excision of a stretch of 18 nt from either the transcribed or nontranscribed DNA strand with the help of the sequence-specific nicking endonucleases Nb.Bpu10I and Nt.Bpu10I. The excised single-stranded oligonucleotide is then swapped for a synthetic …
Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.
8-Oxoguanine (8-oxoG) is a major product of oxidative DNA damage, which induces replication errors and interferes with transcription. By varying the position of single 8-oxoG in a functional gene and manipulating the nucleotide sequence surrounding the lesion, we found that the degree of transcriptional inhibition is independent of the distance from the transcription start or the localization within the transcribed or the non-transcribed DNA strand. However, it is strongly dependent on the sequence context and also proportional to cellular expression of 8-oxoguanine DNA glycosylase (OGG1)-demonstrating that transcriptional arrest does not take place at unrepaired 8-oxoG and proving a causal…
Spontaneous mutagenesis in Csb(m/m)Ogg1⁻(/)⁻ mice is attenuated by dietary resveratrol.
Oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are generated endogenously in apparently all living cells. The defect of the repair of 8-oxoG in Csb m/m Ogg1 ―/― mice results in elevated basal levels of these lesions and increased frequencies of spontaneous mutations, which initiate tumorigenesis in the liver if cell proliferation is stimulated. Here, we describe that the phytoalexin resveratrol, applied either for 7 days per gavage (100 mg/kg body wt) or for 3―9 months in the diet (0.04% ad libitum), reduces the endogenous oxidative DNA base damage in the livers of the Csb m/m Ogg1 ―/― mice by 20―30% (P < 0.01). A small but consistent effect is also observed in the wi…
Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region
Oxidized DNA bases, particularly 7,8-dihydro-8-oxoguanine (8-oxoG), are endogenously generated in cells, being a cause of carcinogenic mutations and possibly interfering with gene expression. We found that expression of an oxidatively damaged plasmid DNA is impaired after delivery into human host cells not only due to decreased retention in the transfected cells, but also due to selective silencing of the damaged reporter gene. To test whether the gene silencing was associated with a specific change of the chromatin structure, we determined the levels of histone modifications related to transcriptional activation (acetylated histones H3 and H4) or repression (methylated K9 and K27 of the hi…
8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1.
The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of trans…
Influences of histone deacetylase inhibitors and resveratrol on DNA repair and chromatin compaction
Accessibility of DNA is a prerequisite for both DNA damage and repair. Therefore, the chromatin structure is expected to have major impact on both processes, with opposite consequences for the stability of the genome. To analyse the influence of chromatin compaction on the generation and repair of various types of DNA modifications, we modulated the global chromatin structure of AS52 Chinese hamster ovary cells and HeLa cells by treatment with either histone deacetylase inhibitors or resveratrol and measured the repair kinetics of (i) pyrimidine dimers induced by ultraviolet B, (ii) oxidised purines generated by photosensitisation and (iii) single-strand breaks induced by H2O2, using an alk…
Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand.
CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3'-OH and 5'-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibi…
Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure
High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have modified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fragment encoding for mouse ornithine decarboxylase (ODC) proline-glutamate-serine-threonine-rich (PEST) sequence in order to target the protein to the proteasomes and achieved an unprecedentedly fast GFP turnover in permanently transfected human cells. As early as 1 h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fo…