A recommended workflow for DNase I footprinting using a capillary electrophoresis genetic analyzer

Sivapragasam, Smitha; Pande, Anuja; Grove, Anne

doi:10.1016/j.ab.2015.04.013

Cited by 11 publications

(16 citation statements)

References 4 publications

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“…Since this fragment of the genome is too large for accurate studies with capillary electrophoresis (CE) [21, 37-39], this sequence was divided into two overlapping fragments, 7348-7798 bp (451 bp) and 7662-122 bp (365 bp).…”

Section: Resultsmentioning

confidence: 99%

Interactions of two large antiviral polyamides with the long control region of HPV16

et al. 2016

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PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5’-W2GW7-3’; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7-2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM to 20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5’-W2GW5GW4-3’ via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicate a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

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Section: Resultsmentioning

confidence: 99%

Interactions of two large antiviral polyamides with the long control region of HPV16

et al. 2016

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“…Approximately 1.0 µl digested sample (diluted in formamide to ensure fluorescence intensity compatible with the analyser) and 1 µl of the 1:10 diluted LIZ 500 standards (ABI, Life Technologies) were brought to 25 µl final volume with formamide. An aliquot of 0.05 ng of undigested DNA (to maintain fluorescence intensity compatible with the analyser) and 0.2 ng of digested DNA was used for fragment analysis (Sivapragasam et al ., ). The samples were boiled for 3 min and loaded on to the ABI 3130 analyzer with the default settings of 1.6 kV injection voltage and 15 s injection time.…”

Section: Methodsmentioning

confidence: 97%

Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage

Sivapragasam

Grove

2016

Molecular Microbiology

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The gene encoding Streptomyces coelicolor xanthine dehydrogenase regulator (XdhR) is divergently oriented from xdhABC, which encodes xanthine dehydrogenase (Xdh). Xdh is required for purine salvage pathways. XdhR was previously shown to repress xdhABC expression. We show that XdhR binds the xdhABC-xdhR intergenic region with high affinity (Kd ∼ 0.5 nM). DNaseI footprinting reveals that this complex formation corresponds to XdhR binding the xdhR gene promoter at two adjacent sites; at higher protein concentrations, protection expands to a region that overlaps the transcriptional and translational start sites of xdhABC. While substrates for Xdh have little effect on DNA binding, GTP and ppGpp dissociate the DNA-XdhR complex. Progression of cells to stationary phase, a condition associated with increased (p)ppGpp production, leads to elevated xdhB expression; in contrast, inhibition of Xdh by allopurinol results in xdhB repression. We propose that XdhR is a direct target of (p)ppGpp, and that expression of xdhABC is upregulated during the stringent response to promote purine salvage pathways, maintain GTP homeostasis and ensure continued (p)ppGpp synthesis. During exponential phase growth, basal levels of xdhABC expression may be achieved by GTP serving as a lower-affinity XdhR ligand.

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“…DNase I footprinting. To identify the specific DNA binding sites for OhrR, DNase I footprinting was carried out (25). Two fluorescently labeled PCR products representing sequences upstream of the ohr (272 bp) and ohrR (280 bp) open reading frames were amplified using B. thailandensis E264 genomic DNA as the template.…”

Section: Methodsmentioning

confidence: 99%

“…Electropherograms were processed using GeneMapper (v4.1) software. Electropherograms representing reaction mixtures containing OhrR were overlaid with the electropherogram obtained from DNase I-digested DNA without protein added (25). Each experiment was repeated at least three times.…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose, longer DNA duplexes encompassing ohrO and ohrRO as well as the flanking sequence were 5= end labeled with 6-carboxyfluorescein (6-FAM). The amplified products were incubated in the absence or presence of the identified OhrR variant, followed by DNase I digestion and analysis of the resulting fragments on a genetic analyzer (25). At a DNA/protein ratio of 1:4, reduced WT OhrR protected a single region upstream of the ohr coding region, extending from positions Ϫ95 to Ϫ57 relative to the translational start, followed by subtle hypersensitive cleavage at position Ϫ56 (Fig.…”

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confidence: 99%

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Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans

2018

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Fatty acid hydroperoxides are involved in host-pathogen interactions. In both plants and mammals, polyunsaturated fatty acids are liberated during infection and enzymatically oxidized to the corresponding toxic hydroperoxides during the defensive oxidative burst that is designed to thwart the infection. The bacterial transcription factor OhrR (organic hydroperoxide reductase regulator) is oxidized by organic hydroperoxides, as a result of which the gene encoding organic hydroperoxide reductase is induced. This enzyme converts the hydroperoxides to less toxic alcohols. We show here that OhrR from represses expression of Gene expression is induced by cumene hydroperoxide and to a lesser extent by inorganic oxidants; however, Ohr contributes to degradation only of the organic hydroperoxide. OhrR, which binds specific sites in both and promoters, as evidenced by DNase I footprinting, belongs to the 2-Cys subfamily of OhrR proteins, and its oxidation leads to reversible disulfide bond formation between conserved N- and C-terminal cysteines in separate monomers. Oxidation of the N-terminal Cys is sufficient for attenuation of DNA binding , with complete restoration of DNA binding occurring on addition of a reducing agent. Surprisingly, both overexpression of and deletion of results in enhanced survival on exposure to organic hydroperoxide While Δ cells are more virulent in a model of infection, Δ cells are less so. Taken together, our data suggest that OhrR has several unconventional features and that both OhrR and organic hydroperoxides may contribute to virulence.

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A recommended workflow for DNase I footprinting using a capillary electrophoresis genetic analyzer

Cited by 11 publications

References 4 publications

Interactions of two large antiviral polyamides with the long control region of HPV16

Interactions of two large antiviral polyamides with the long control region of HPV16

Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage

Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans

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