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Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.

Allgayer J, Kitsera N, von der Lippen C, Epe B, Khobta A - Nucleic Acids Res. (2013)

Bottom Line: 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 connection between 8-oxoG excision and the inhibition of transcription.This anticipation was fully confirmed by direct biochemical assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany.

ABSTRACT
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 connection between 8-oxoG excision and the inhibition of transcription. We identified the 5'-CAGGGC[8-oxoG]GACTG-3' motif as having only minimal transcription-inhibitory potential in cells, based on which we predicted that 8-oxoG excision is particularly inefficient in this sequence context. This anticipation was fully confirmed by direct biochemical assays. Furthermore, in DNA containing a bistranded Cp[8-oxoG]/Cp[8-oxoG] clustered lesion, the excision rates differed between the two strands at least by a factor of 9, clearly demonstrating that the excision preference is defined by the DNA strand asymmetry rather than the overall geometry of the double helix or local duplex stability.

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Effect of single 8-oxoG in the 5′-UTR on expression of the EGFP-coding gene in Hela cells. (A) Agarose gel analyses of vector DNA harbouring synthetic 18mer oligonucleotides containing either G or 8-oxoG. The aliquots were incubated with Fpg DNA glycosylase to detect 8-oxoG by conversion of covalently closed DNA (cc) to open circles (oc). Diagrams illustrate position of 8-oxoG (star) with respect to transcription start (broken arrow) and the protein-coding DNA sequence (arrow). (B–F) Flow cytometric analyses of HeLa cells co-transfected with equal amounts of a vector encoding for DsRed-Monomer and the indicated EGFP encoding plasmids. (B) The principle of quantitative analysis of EGFP expression in transfected cells. (C and E) Results of typical experiments. (D and F) Relative EGFP expression summarized for two independently prepared 8-oxoG/G substrate pairs (separate bars, each representing mean of three transfection experiments±SD). See also Supplementary Figure S2.
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gkt620-F1: Effect of single 8-oxoG in the 5′-UTR on expression of the EGFP-coding gene in Hela cells. (A) Agarose gel analyses of vector DNA harbouring synthetic 18mer oligonucleotides containing either G or 8-oxoG. The aliquots were incubated with Fpg DNA glycosylase to detect 8-oxoG by conversion of covalently closed DNA (cc) to open circles (oc). Diagrams illustrate position of 8-oxoG (star) with respect to transcription start (broken arrow) and the protein-coding DNA sequence (arrow). (B–F) Flow cytometric analyses of HeLa cells co-transfected with equal amounts of a vector encoding for DsRed-Monomer and the indicated EGFP encoding plasmids. (B) The principle of quantitative analysis of EGFP expression in transfected cells. (C and E) Results of typical experiments. (D and F) Relative EGFP expression summarized for two independently prepared 8-oxoG/G substrate pairs (separate bars, each representing mean of three transfection experiments±SD). See also Supplementary Figure S2.

Mentions: We constructed plasmid vectors, suitable for incorporation of synthetic oligonucleotides into the 5′- UTR of the EGFP gene and into the 3′-UTR, by inserting an arbitrarily chosen DNA sequence flanked by two tandemly located sites for the Nb.BsrDI nicking endonuclease. For both locations, the new sequences were placed in alternative orientations (Supplementary Figure S1). Vectors containing the modified 5′-UTR will be further referred to as pZAJ-5w and pZAJ-5c. Similarly, the vectors with modified 3′-UTR were named pZAJ-3w and pZAJ-3c. Tandem Nb.BsrDI sites of each of the four vectors were used to swap the enclosed 18-nt fragment of the native DNA strand for a synthetic deoxyribo-oligonucleotide, according to the principle described previously (31). The oligonucleotide contained single 8-oxoG in a 5′-A[8-oxoG]C context (Supplementary Table S1). Successful incorporation of 8-oxoG was confirmed by analytical incision of the obtained hybrid molecules with Fpg (Figure 1A and Supplementary Figure S2). The vectors obtained by insertion of unmodified oligonucleotides were only partly converted to the open circular form by incision at a small number of Fpg-sensitive sites intrinsically present in the plasmid DNA. In contrast, the vectors harbouring the 8-oxoG containing oligonucleotide were fully incised, thereby indicating that all molecules contained the synthetic 8-oxoG in addition to the basal non-specific DNA damage.Figure 1.


Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.

Allgayer J, Kitsera N, von der Lippen C, Epe B, Khobta A - Nucleic Acids Res. (2013)

Effect of single 8-oxoG in the 5′-UTR on expression of the EGFP-coding gene in Hela cells. (A) Agarose gel analyses of vector DNA harbouring synthetic 18mer oligonucleotides containing either G or 8-oxoG. The aliquots were incubated with Fpg DNA glycosylase to detect 8-oxoG by conversion of covalently closed DNA (cc) to open circles (oc). Diagrams illustrate position of 8-oxoG (star) with respect to transcription start (broken arrow) and the protein-coding DNA sequence (arrow). (B–F) Flow cytometric analyses of HeLa cells co-transfected with equal amounts of a vector encoding for DsRed-Monomer and the indicated EGFP encoding plasmids. (B) The principle of quantitative analysis of EGFP expression in transfected cells. (C and E) Results of typical experiments. (D and F) Relative EGFP expression summarized for two independently prepared 8-oxoG/G substrate pairs (separate bars, each representing mean of three transfection experiments±SD). See also Supplementary Figure S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794583&req=5

gkt620-F1: Effect of single 8-oxoG in the 5′-UTR on expression of the EGFP-coding gene in Hela cells. (A) Agarose gel analyses of vector DNA harbouring synthetic 18mer oligonucleotides containing either G or 8-oxoG. The aliquots were incubated with Fpg DNA glycosylase to detect 8-oxoG by conversion of covalently closed DNA (cc) to open circles (oc). Diagrams illustrate position of 8-oxoG (star) with respect to transcription start (broken arrow) and the protein-coding DNA sequence (arrow). (B–F) Flow cytometric analyses of HeLa cells co-transfected with equal amounts of a vector encoding for DsRed-Monomer and the indicated EGFP encoding plasmids. (B) The principle of quantitative analysis of EGFP expression in transfected cells. (C and E) Results of typical experiments. (D and F) Relative EGFP expression summarized for two independently prepared 8-oxoG/G substrate pairs (separate bars, each representing mean of three transfection experiments±SD). See also Supplementary Figure S2.
Mentions: We constructed plasmid vectors, suitable for incorporation of synthetic oligonucleotides into the 5′- UTR of the EGFP gene and into the 3′-UTR, by inserting an arbitrarily chosen DNA sequence flanked by two tandemly located sites for the Nb.BsrDI nicking endonuclease. For both locations, the new sequences were placed in alternative orientations (Supplementary Figure S1). Vectors containing the modified 5′-UTR will be further referred to as pZAJ-5w and pZAJ-5c. Similarly, the vectors with modified 3′-UTR were named pZAJ-3w and pZAJ-3c. Tandem Nb.BsrDI sites of each of the four vectors were used to swap the enclosed 18-nt fragment of the native DNA strand for a synthetic deoxyribo-oligonucleotide, according to the principle described previously (31). The oligonucleotide contained single 8-oxoG in a 5′-A[8-oxoG]C context (Supplementary Table S1). Successful incorporation of 8-oxoG was confirmed by analytical incision of the obtained hybrid molecules with Fpg (Figure 1A and Supplementary Figure S2). The vectors obtained by insertion of unmodified oligonucleotides were only partly converted to the open circular form by incision at a small number of Fpg-sensitive sites intrinsically present in the plasmid DNA. In contrast, the vectors harbouring the 8-oxoG containing oligonucleotide were fully incised, thereby indicating that all molecules contained the synthetic 8-oxoG in addition to the basal non-specific DNA damage.Figure 1.

Bottom Line: 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 connection between 8-oxoG excision and the inhibition of transcription.This anticipation was fully confirmed by direct biochemical assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany.

ABSTRACT
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 connection between 8-oxoG excision and the inhibition of transcription. We identified the 5'-CAGGGC[8-oxoG]GACTG-3' motif as having only minimal transcription-inhibitory potential in cells, based on which we predicted that 8-oxoG excision is particularly inefficient in this sequence context. This anticipation was fully confirmed by direct biochemical assays. Furthermore, in DNA containing a bistranded Cp[8-oxoG]/Cp[8-oxoG] clustered lesion, the excision rates differed between the two strands at least by a factor of 9, clearly demonstrating that the excision preference is defined by the DNA strand asymmetry rather than the overall geometry of the double helix or local duplex stability.

Show MeSH
Related in: MedlinePlus