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[56] F o o t p r i n t i n g P r o t e i n - D N A C o m p l e x e s W i t h T-Rays By JEFFREY J. HAYES, LAUaAr~CE KAM, and THOMAS D. TULLIUS Introduction
Proteins that bind to specific sequences of DNA are important components of gene regulatory systems. Protein-DNA complexes are commonly studied by in vitro techniques such as DNase I footprintingI and methylation interference: Another innovative technique uses UV light to create photochemical lesions in DNA, which are susceptible to later chemical cleavage) Bound proteins affect the yield of these photoproducts, giving rise to "photofootprints." Each of these techniques, however, suffers from the inherent DNA base or sequence specificity of the probe used. A reagent system consisting of [Fe(II)EDTA] z-, hydrogen peroxide, and sodium ascorbate has been shown to cleave DNA with little sequence specificity, thus allowing investigation of protein contact with every DNA sequence position/,5 The cleavage reagent in this system is likely the hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II). A key advantage of the hydroxyl radical is that its small size (with respect to the protein-DNA system of interest) results in very highresolution footprints. This footprinting method is not applicable in all solution conditions, though, and it requires the addition of several reagents to a DNA-protein sample that may interfere with the function of the DNA-binding protein, or otherwise be inconvenient) We describe here an analog of the recently introduced method of hydroxyl radical footprinting. 4,5 We have duplicated the high-resolution footprints of this technique, while substituting y-radiation for the chemical reagents used heretofore, y-Radiation has previously been shown to mediate DNA cleavage with no apparent base or sequence specificity.6 The primary cutting reagent, as in the analogous chemical reagent system, is thought to be the hydroxyl radical. 7 This new method requires no addition of reagents to the protein-DNA binding solution. 1 D. J. Galas and A. Schmitz, Nucleic Acids Res. 5, 3157 (1978). 2 U. Siebenlist and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 77, 122 (1980). 3 M. M. Becker and J. C. Wang, Nature (London) 309, 682 (1984). 4 T. D. Tullius and B. A. Dombroski, Proc. Natl. Acad. Sci. U.S.A. 83, 5469 (1986). 5 T. D. Tullius, B. A. Dombroski, M. E. A. Churchill, and L. Kam, this series, Vol. 155, p. 537. 6 W. D. Henner, S. M. Grunberg, and W. A. Haseltine, J. Biol. Chem. 257, 11750 (1982). 7 T. D. Tullius and B. A. Dombroski, Science 230, 679 (1985).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, inc. All rights of reproduction in any form reserved.
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FIG. 1. Comparison of [Fe(II)EDTA] 2 and y-ray footprinting reagents. (a) Autoradiograph of denaturing polyacrylamide electrophoresis gel on which was separated the DNA products from the [Fe(II)EDTA] 2- and y-ray footprinting reactions: lane 1, Maxam-Gilbert
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Materials and Methods Footprinting of the bacteriophage )~ repressor/operator complex with the [Fe(II)EDTA] 2- reagent system has been described in detailJ ,5 y-Ray footprinting is carried out in the same manner and under the same conditions, except that the volume of the DNA-protein binding solution is larger. This increases the effective target volume for the radiation and thus increases the rate of production of hydroxyl radicals. Typically, 20 fmol of 32P-end-labeled DNA is dissolved in 180/.d of repressor binding buffer [10 m M Bis-Tris-HC1 (pH 7.0), 50 mM KCI, 1 m M CaCl2]. ~ repressor (20/.tl, 12.5 pmol) (a generous gift from M. Brenowitz and G. Ackers), diluted in the same buffer, is added, and the mixture is incubated at room temperature for 15 min. The sample is irradiated at a dose rate of approximately 5000 rads/min for 7 min at room temperature in a Shepherd 137Cs irradiator. Control DNA samples (without added protein) are irradiated for 3 min because DNA damage accumulates more quickly in these samples. The total dose is empirically determined, and it varies as a function of sample volume, temperature, and protein and glycerol content. The irradiated samples are analyzed by denaturing gel electrophoresis, autoradiography, and densitometry, as described) Comments We first obtained cleavage patterns for a 692-base pair restriction fragment containing the OR1 operator site, using both y-radiation and the [Fe(II)EDTA] 2- reagent system. Conventional strategy was followed in that the fragment was uniquely end-labeled prior to the cleavage experiment. 4,5 The patterns of cleavage obtained by the two methods are shown in Fig. la, lanes 2 and 3. As has been discussed before, 7 the cutting in the [Fe(II)EDTA]2--treated sample occurs to nearly an equal extent at each base position, resulting in a cleavage pattern which is not directly a function of DNA sequence. The cutting pattern of the y-ray-treated sample also is evenly distributed over all of the nucleotide positions and is nearly identical to that observed with the [Fe(II)EDTA] 2- method.
G-specific reaction; fanes 2 and 3, [Fe(II)EDTA] 2- and y-ray cleavage, respectively, of naked DNA control; lanes 4 and 5, [Fe(II)EDTA] 2- and y-ray footprinting of ~ repressor/ operator complex, respectively, The arrow points to the central base in the operator sequence Oal, and the bar spans the 17 bases of the operator site. (b) Densitometer scans of the gel shown in Fig. la. Scans are numbered corresponding to the lanes in Fig. la. The arrow points to the central position (the dyad) in the 17 base-pair-long operator sequence.
548
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
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A closer inspection of the densitometer scans (Fig. Ib) reveals that even the subtle differences in cleavage frequency at each position found in the [Fe(II)EDTA]2--treated sample are exactly reproduced in the y-ray-treated sample. These small fluctuations are thought to be due to sequence-dependent conformational variation in the DNA helix, 7 most likely the width of the minor groove. 8 This result is somewhat surprising since the point of origin of the reactive species produced by ionizing radiation is not constrained to be some distance away from the DNA helix as in the [Fe(II)EDTA] 2- method. 9 It therefore appears that the majority of hydroxyl radical flux resulting in DNA backbone cleavage in both cases comes from the bulk solvent, and that sensitivity to structural irregularities in the helix is not lost by the use of y-radiation as a source of hydroxyl radicals. This result suggests that water molecules bound in the grooves of the DNA helix (which could be homolytically cleaved by the highenergy radiation and subsequently react with the DNA backbone) contribute very little to the cutting pattern in the y-ray-treated sample. The advantages of the hydroxyl radical as a footprinting reagent have been previously discussed.I° The [Fe(II)EDTA] 2- system has been used to demonstrate clearly that the h repressor binds to one side of an OR 1containing DNA molecule. 4 Thus, this system is well suited to test the use of y-radiation as a footprinting reagent. The amount of radiation used was empirically determined, since the extent of DNA damage by radiation is influenced by salt conditions 1~ and the presence of radioprotectants (such as proteins). Because of the nature of the experiment, the goal is to create no more than one strand break per molecule. The pattern of DNA fragments which results from hydroxyl radical cleavage of the repressor-DNA complex (Fig. la,b, lanes 4 and 5) is clearly different from the pattern of the controls (no repressor added, Fig. la,b, lanes 2 and 3) and is qualitatively similar to that observed in the earlier study. 4 Both the [Fe(II)EDTA] 2- system and y-radiation give very similar footprinting patterns. The presence of nucleotides that are readil~¢ cleaved between sites of protection leads directly to the conclusion that repressor binds to one side of the DNA molecule, as shown by X-ray crystallography of a cocrystal of the repressor-DNA complex. 12
s T. D. Tuilius and A. M. Burkhoffl in "Structure and Expression, Volume 3: DNA Bending and Curvature" (W. K. Olson, M. H. Sarma, R. H. Sarma, and M. Sundaralingam, eds.), p.77. Adenine Press, Guilderland, New York, 1988. 9 T. D. Tullius, Trends Biochem. Sci. 12, 297 (1987). to T. D. Tullius, Nature (London) 332, 663 (1988). 11 j. Ward and I. Kuo, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 18, 381 (1970). 12 S. R. Jordan and C. O. Pabo, Science 242, 893 (1988).
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An identical result is obtained whether the OR 1 site is contained in a small linear DNA fragment or in a form I plasmid during the footprinting experiment (data not shown). Perspectives We have shown here that y-radiation is a suitable alternative to chemical systems for generating hydroxyl radicals for footprinting purposes. The technique can be used to carry out footprinting in protein-DNAbinding solutions that are not conducive to the use of the [Fe(II)EDTA] 2reagent system, such as those that contain moderate amounts of glycerol, 5 or those that cannot tolerate the addition of one of the reagent components, such as hydrogen peroxide. 5,~3 Morever, the ability of y-radiation to penetrate matter suggests that extention of the technique to in vivo systems might be possible. Such experiments could be performed on a model system constructed on a plasmid or could be applied to interactions of proteins with chromosomal DNA when used in combination with an appropriate detection scheme. 14 An in vivo application would offer the opportunity of determining highresolution footprints of proteins bound to DNA inside ceils. On the debit side, the delivery of large radiation doses to an organism causes a great deal of collateral damage, which over an extended period of time may not be acceptable. This problem may be rectified by the use of particle accelerators, such as those in use for pulse radiolysis experiments, to generate the required radiation dose in the shortest time period possible. Acknowledgments This research was supported by grants from the American Cancer Society (Institutional Research Grant IN-11Z) and the National Cancer Institute of the National Institutes of Health (CA 37444). T.D.T. is a fellow of the Alfred P. Sloan Foundation, a Camille and Henry Dreyfus Teacher-Scholar, and the recipient of a Research Career Development Award from the National Cancer Institute (CA 01208).
13 K. E. Vrana, M. E. A. Churchill, T. D. Tullius, and D. D. Brown, Mol. Cell. Biol. 8, 1684
(1988). 14 G. M. Church and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 81, 1991 (1984).
y-aAV FOOTPRINTING
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[56] F o o t p r i n t i n g P r o t e i n - D N A C o m p l e x e s W i t h T-Rays By JEFFREY J. HAYES, LAUaAr~CE KAM, and THOMAS D. TULLIUS Introduction
Proteins that bind to specific sequences of DNA are important components of gene regulatory systems. Protein-DNA complexes are commonly studied by in vitro techniques such as DNase I footprintingI and methylation interference: Another innovative technique uses UV light to create photochemical lesions in DNA, which are susceptible to later chemical cleavage) Bound proteins affect the yield of these photoproducts, giving rise to "photofootprints." Each of these techniques, however, suffers from the inherent DNA base or sequence specificity of the probe used. A reagent system consisting of [Fe(II)EDTA] z-, hydrogen peroxide, and sodium ascorbate has been shown to cleave DNA with little sequence specificity, thus allowing investigation of protein contact with every DNA sequence position/,5 The cleavage reagent in this system is likely the hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II). A key advantage of the hydroxyl radical is that its small size (with respect to the protein-DNA system of interest) results in very highresolution footprints. This footprinting method is not applicable in all solution conditions, though, and it requires the addition of several reagents to a DNA-protein sample that may interfere with the function of the DNA-binding protein, or otherwise be inconvenient) We describe here an analog of the recently introduced method of hydroxyl radical footprinting. 4,5 We have duplicated the high-resolution footprints of this technique, while substituting y-radiation for the chemical reagents used heretofore, y-Radiation has previously been shown to mediate DNA cleavage with no apparent base or sequence specificity.6 The primary cutting reagent, as in the analogous chemical reagent system, is thought to be the hydroxyl radical. 7 This new method requires no addition of reagents to the protein-DNA binding solution. 1 D. J. Galas and A. Schmitz, Nucleic Acids Res. 5, 3157 (1978). 2 U. Siebenlist and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 77, 122 (1980). 3 M. M. Becker and J. C. Wang, Nature (London) 309, 682 (1984). 4 T. D. Tullius and B. A. Dombroski, Proc. Natl. Acad. Sci. U.S.A. 83, 5469 (1986). 5 T. D. Tullius, B. A. Dombroski, M. E. A. Churchill, and L. Kam, this series, Vol. 155, p. 537. 6 W. D. Henner, S. M. Grunberg, and W. A. Haseltine, J. Biol. Chem. 257, 11750 (1982). 7 T. D. Tullius and B. A. Dombroski, Science 230, 679 (1985).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, inc. All rights of reproduction in any form reserved.
546
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FIG. 1. Comparison of [Fe(II)EDTA] 2 and y-ray footprinting reagents. (a) Autoradiograph of denaturing polyacrylamide electrophoresis gel on which was separated the DNA products from the [Fe(II)EDTA] 2- and y-ray footprinting reactions: lane 1, Maxam-Gilbert
[56]
y-RAY FOOTPRINTING
547
Materials and Methods Footprinting of the bacteriophage )~ repressor/operator complex with the [Fe(II)EDTA] 2- reagent system has been described in detailJ ,5 y-Ray footprinting is carried out in the same manner and under the same conditions, except that the volume of the DNA-protein binding solution is larger. This increases the effective target volume for the radiation and thus increases the rate of production of hydroxyl radicals. Typically, 20 fmol of 32P-end-labeled DNA is dissolved in 180/.d of repressor binding buffer [10 m M Bis-Tris-HC1 (pH 7.0), 50 mM KCI, 1 m M CaCl2]. ~ repressor (20/.tl, 12.5 pmol) (a generous gift from M. Brenowitz and G. Ackers), diluted in the same buffer, is added, and the mixture is incubated at room temperature for 15 min. The sample is irradiated at a dose rate of approximately 5000 rads/min for 7 min at room temperature in a Shepherd 137Cs irradiator. Control DNA samples (without added protein) are irradiated for 3 min because DNA damage accumulates more quickly in these samples. The total dose is empirically determined, and it varies as a function of sample volume, temperature, and protein and glycerol content. The irradiated samples are analyzed by denaturing gel electrophoresis, autoradiography, and densitometry, as described) Comments We first obtained cleavage patterns for a 692-base pair restriction fragment containing the OR1 operator site, using both y-radiation and the [Fe(II)EDTA] 2- reagent system. Conventional strategy was followed in that the fragment was uniquely end-labeled prior to the cleavage experiment. 4,5 The patterns of cleavage obtained by the two methods are shown in Fig. la, lanes 2 and 3. As has been discussed before, 7 the cutting in the [Fe(II)EDTA]2--treated sample occurs to nearly an equal extent at each base position, resulting in a cleavage pattern which is not directly a function of DNA sequence. The cutting pattern of the y-ray-treated sample also is evenly distributed over all of the nucleotide positions and is nearly identical to that observed with the [Fe(II)EDTA] 2- method.
G-specific reaction; fanes 2 and 3, [Fe(II)EDTA] 2- and y-ray cleavage, respectively, of naked DNA control; lanes 4 and 5, [Fe(II)EDTA] 2- and y-ray footprinting of ~ repressor/ operator complex, respectively, The arrow points to the central base in the operator sequence Oal, and the bar spans the 17 bases of the operator site. (b) Densitometer scans of the gel shown in Fig. la. Scans are numbered corresponding to the lanes in Fig. la. The arrow points to the central position (the dyad) in the 17 base-pair-long operator sequence.
548
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[56l
A closer inspection of the densitometer scans (Fig. Ib) reveals that even the subtle differences in cleavage frequency at each position found in the [Fe(II)EDTA]2--treated sample are exactly reproduced in the y-ray-treated sample. These small fluctuations are thought to be due to sequence-dependent conformational variation in the DNA helix, 7 most likely the width of the minor groove. 8 This result is somewhat surprising since the point of origin of the reactive species produced by ionizing radiation is not constrained to be some distance away from the DNA helix as in the [Fe(II)EDTA] 2- method. 9 It therefore appears that the majority of hydroxyl radical flux resulting in DNA backbone cleavage in both cases comes from the bulk solvent, and that sensitivity to structural irregularities in the helix is not lost by the use of y-radiation as a source of hydroxyl radicals. This result suggests that water molecules bound in the grooves of the DNA helix (which could be homolytically cleaved by the highenergy radiation and subsequently react with the DNA backbone) contribute very little to the cutting pattern in the y-ray-treated sample. The advantages of the hydroxyl radical as a footprinting reagent have been previously discussed.I° The [Fe(II)EDTA] 2- system has been used to demonstrate clearly that the h repressor binds to one side of an OR 1containing DNA molecule. 4 Thus, this system is well suited to test the use of y-radiation as a footprinting reagent. The amount of radiation used was empirically determined, since the extent of DNA damage by radiation is influenced by salt conditions 1~ and the presence of radioprotectants (such as proteins). Because of the nature of the experiment, the goal is to create no more than one strand break per molecule. The pattern of DNA fragments which results from hydroxyl radical cleavage of the repressor-DNA complex (Fig. la,b, lanes 4 and 5) is clearly different from the pattern of the controls (no repressor added, Fig. la,b, lanes 2 and 3) and is qualitatively similar to that observed in the earlier study. 4 Both the [Fe(II)EDTA] 2- system and y-radiation give very similar footprinting patterns. The presence of nucleotides that are readil~¢ cleaved between sites of protection leads directly to the conclusion that repressor binds to one side of the DNA molecule, as shown by X-ray crystallography of a cocrystal of the repressor-DNA complex. 12
s T. D. Tuilius and A. M. Burkhoffl in "Structure and Expression, Volume 3: DNA Bending and Curvature" (W. K. Olson, M. H. Sarma, R. H. Sarma, and M. Sundaralingam, eds.), p.77. Adenine Press, Guilderland, New York, 1988. 9 T. D. Tullius, Trends Biochem. Sci. 12, 297 (1987). to T. D. Tullius, Nature (London) 332, 663 (1988). 11 j. Ward and I. Kuo, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 18, 381 (1970). 12 S. R. Jordan and C. O. Pabo, Science 242, 893 (1988).
[56]
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549
An identical result is obtained whether the OR 1 site is contained in a small linear DNA fragment or in a form I plasmid during the footprinting experiment (data not shown). Perspectives We have shown here that y-radiation is a suitable alternative to chemical systems for generating hydroxyl radicals for footprinting purposes. The technique can be used to carry out footprinting in protein-DNAbinding solutions that are not conducive to the use of the [Fe(II)EDTA] 2reagent system, such as those that contain moderate amounts of glycerol, 5 or those that cannot tolerate the addition of one of the reagent components, such as hydrogen peroxide. 5,~3 Morever, the ability of y-radiation to penetrate matter suggests that extention of the technique to in vivo systems might be possible. Such experiments could be performed on a model system constructed on a plasmid or could be applied to interactions of proteins with chromosomal DNA when used in combination with an appropriate detection scheme. 14 An in vivo application would offer the opportunity of determining highresolution footprints of proteins bound to DNA inside ceils. On the debit side, the delivery of large radiation doses to an organism causes a great deal of collateral damage, which over an extended period of time may not be acceptable. This problem may be rectified by the use of particle accelerators, such as those in use for pulse radiolysis experiments, to generate the required radiation dose in the shortest time period possible. Acknowledgments This research was supported by grants from the American Cancer Society (Institutional Research Grant IN-11Z) and the National Cancer Institute of the National Institutes of Health (CA 37444). T.D.T. is a fellow of the Alfred P. Sloan Foundation, a Camille and Henry Dreyfus Teacher-Scholar, and the recipient of a Research Career Development Award from the National Cancer Institute (CA 01208).
13 K. E. Vrana, M. E. A. Churchill, T. D. Tullius, and D. D. Brown, Mol. Cell. Biol. 8, 1684
(1988). 14 G. M. Church and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 81, 1991 (1984).
