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A robust assay to measure DNA topology-dependent protein binding affinity.

Litwin TR, Solà M, Holt IJ, Neuman KC - Nucleic Acids Res. (2014)

Bottom Line: A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques.Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number.Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA.

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Affiliation: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA Mitochondrial Biology Unit, Medical Research Council, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.

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(A) Relative binding affinities (Ka) normalized to the affinity for topoisomer ΔLk = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer ΔLk = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of ΔLk = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with ΔLk values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative Ka value for ΔLk = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing ΔLk. The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids (KaN/Ka0) and for highly supercoiled (ΔLk = −23) plasmids (KaS/Ka0).
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Figure 3: (A) Relative binding affinities (Ka) normalized to the affinity for topoisomer ΔLk = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer ΔLk = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of ΔLk = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with ΔLk values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative Ka value for ΔLk = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing ΔLk. The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids (KaN/Ka0) and for highly supercoiled (ΔLk = −23) plasmids (KaS/Ka0).

Mentions: We next measured the topology-dependent affinity of several different DNA binding proteins to determine if there are commonalities among different classes of enzymes. We tested catalytic mutants of two type IB topoisomerases (human nuclear topoisomerase IB, ntop1; and human mitochondrial topoisomerase IB, top1mt), a prokaryotic helicase (RecQ), a restriction endonuclease (EcoRV) and a mitochondrial transcription factor (Tfam) (Figure 3A). In general, the proteins displayed higher or equal binding affinity for negatively supercoiled compared to relaxed DNA, but the degree of topology-dependent binding varied among the proteins (Figure 3A).


A robust assay to measure DNA topology-dependent protein binding affinity.

Litwin TR, Solà M, Holt IJ, Neuman KC - Nucleic Acids Res. (2014)

(A) Relative binding affinities (Ka) normalized to the affinity for topoisomer ΔLk = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer ΔLk = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of ΔLk = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with ΔLk values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative Ka value for ΔLk = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing ΔLk. The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids (KaN/Ka0) and for highly supercoiled (ΔLk = −23) plasmids (KaS/Ka0).
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Figure 3: (A) Relative binding affinities (Ka) normalized to the affinity for topoisomer ΔLk = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer ΔLk = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of ΔLk = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with ΔLk values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative Ka value for ΔLk = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing ΔLk. The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids (KaN/Ka0) and for highly supercoiled (ΔLk = −23) plasmids (KaS/Ka0).
Mentions: We next measured the topology-dependent affinity of several different DNA binding proteins to determine if there are commonalities among different classes of enzymes. We tested catalytic mutants of two type IB topoisomerases (human nuclear topoisomerase IB, ntop1; and human mitochondrial topoisomerase IB, top1mt), a prokaryotic helicase (RecQ), a restriction endonuclease (EcoRV) and a mitochondrial transcription factor (Tfam) (Figure 3A). In general, the proteins displayed higher or equal binding affinity for negatively supercoiled compared to relaxed DNA, but the degree of topology-dependent binding varied among the proteins (Figure 3A).

Bottom Line: A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques.Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number.Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA.

View Article: PubMed Central - PubMed

Affiliation: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA Mitochondrial Biology Unit, Medical Research Council, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.

Show MeSH
Related in: MedlinePlus