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The guanine-quadruplex structure in the human c-myc gene's promoter is converted into B-DNA form by the human poly(ADP-ribose)polymerase-1.

Fekete A, Kenesi E, Hunyadi-Gulyas E, Durgo H, Berko B, Dunai ZA, Bauer PI - PLoS ONE (2012)

Bottom Line: We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form.The first Zn-finger structure present in h PARP-1 participates in this interaction.PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.

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In vitro effect of wild type h c-myc GQ structure on the enzymatic activity of h PARP-1.PARP-1 (1 pmol) was incubated with 6-biotin-17-NAD (75 µM) in the presence of various concentrations of c-myc GQ oligonucleotides for 10 minutes at 23°C. At the end of incubation an equal volume of Laemmli sample buffer was admixed, boiled samples were electrophoresed (10% SDS-PAGE), and transblotted onto nitrocellulose sheet and the aspecific binding sites were blocked by incubating the blot in a solution containing 3% fat free milk dissolved in PBS. This was followed by an incubation of the blot with HPO conjugated streptavidine (1 µg/ml) for an hour. Bound biotin-ADP-ribose was detected by ECL. The fluorogram shown in the insert (c-myc GQ concentrations used in the assays are from left to right: 0, 5, 10, 15 and 25 µM). Spot intensities were quantitated using a Bio-Rad gel-analyzer system. Ordinate shows the incorporation of biotin-ADP-ribose in pixel intensity/pmol PARP-1× min units while the applied GQ concentrations are shown on the abscissa. One representative experiment is shown from three independent experiments.
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pone-0042690-g002: In vitro effect of wild type h c-myc GQ structure on the enzymatic activity of h PARP-1.PARP-1 (1 pmol) was incubated with 6-biotin-17-NAD (75 µM) in the presence of various concentrations of c-myc GQ oligonucleotides for 10 minutes at 23°C. At the end of incubation an equal volume of Laemmli sample buffer was admixed, boiled samples were electrophoresed (10% SDS-PAGE), and transblotted onto nitrocellulose sheet and the aspecific binding sites were blocked by incubating the blot in a solution containing 3% fat free milk dissolved in PBS. This was followed by an incubation of the blot with HPO conjugated streptavidine (1 µg/ml) for an hour. Bound biotin-ADP-ribose was detected by ECL. The fluorogram shown in the insert (c-myc GQ concentrations used in the assays are from left to right: 0, 5, 10, 15 and 25 µM). Spot intensities were quantitated using a Bio-Rad gel-analyzer system. Ordinate shows the incorporation of biotin-ADP-ribose in pixel intensity/pmol PARP-1× min units while the applied GQ concentrations are shown on the abscissa. One representative experiment is shown from three independent experiments.

Mentions: The 50% inhibitory concentration of TMPyP4 was around 8–12 µM (Fig. 1A). The PARP-1 bound c-myc GQ increased the enzymatic activity of the protein in a concentration-dependent way and GQ was a better activator of the enzyme than single-stranded DNA or RNA (Fig. 2 and Table S1) [26]. The c-myc GQ concentration resulting in 50% activation of the enzymatic activity PARP-1 enzymatic activity was around 10 µM (Fig. 2).


The guanine-quadruplex structure in the human c-myc gene's promoter is converted into B-DNA form by the human poly(ADP-ribose)polymerase-1.

Fekete A, Kenesi E, Hunyadi-Gulyas E, Durgo H, Berko B, Dunai ZA, Bauer PI - PLoS ONE (2012)

In vitro effect of wild type h c-myc GQ structure on the enzymatic activity of h PARP-1.PARP-1 (1 pmol) was incubated with 6-biotin-17-NAD (75 µM) in the presence of various concentrations of c-myc GQ oligonucleotides for 10 minutes at 23°C. At the end of incubation an equal volume of Laemmli sample buffer was admixed, boiled samples were electrophoresed (10% SDS-PAGE), and transblotted onto nitrocellulose sheet and the aspecific binding sites were blocked by incubating the blot in a solution containing 3% fat free milk dissolved in PBS. This was followed by an incubation of the blot with HPO conjugated streptavidine (1 µg/ml) for an hour. Bound biotin-ADP-ribose was detected by ECL. The fluorogram shown in the insert (c-myc GQ concentrations used in the assays are from left to right: 0, 5, 10, 15 and 25 µM). Spot intensities were quantitated using a Bio-Rad gel-analyzer system. Ordinate shows the incorporation of biotin-ADP-ribose in pixel intensity/pmol PARP-1× min units while the applied GQ concentrations are shown on the abscissa. One representative experiment is shown from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412819&req=5

pone-0042690-g002: In vitro effect of wild type h c-myc GQ structure on the enzymatic activity of h PARP-1.PARP-1 (1 pmol) was incubated with 6-biotin-17-NAD (75 µM) in the presence of various concentrations of c-myc GQ oligonucleotides for 10 minutes at 23°C. At the end of incubation an equal volume of Laemmli sample buffer was admixed, boiled samples were electrophoresed (10% SDS-PAGE), and transblotted onto nitrocellulose sheet and the aspecific binding sites were blocked by incubating the blot in a solution containing 3% fat free milk dissolved in PBS. This was followed by an incubation of the blot with HPO conjugated streptavidine (1 µg/ml) for an hour. Bound biotin-ADP-ribose was detected by ECL. The fluorogram shown in the insert (c-myc GQ concentrations used in the assays are from left to right: 0, 5, 10, 15 and 25 µM). Spot intensities were quantitated using a Bio-Rad gel-analyzer system. Ordinate shows the incorporation of biotin-ADP-ribose in pixel intensity/pmol PARP-1× min units while the applied GQ concentrations are shown on the abscissa. One representative experiment is shown from three independent experiments.
Mentions: The 50% inhibitory concentration of TMPyP4 was around 8–12 µM (Fig. 1A). The PARP-1 bound c-myc GQ increased the enzymatic activity of the protein in a concentration-dependent way and GQ was a better activator of the enzyme than single-stranded DNA or RNA (Fig. 2 and Table S1) [26]. The c-myc GQ concentration resulting in 50% activation of the enzymatic activity PARP-1 enzymatic activity was around 10 µM (Fig. 2).

Bottom Line: We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form.The first Zn-finger structure present in h PARP-1 participates in this interaction.PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.

Show MeSH
Related in: MedlinePlus