328
APPLICATIONS
[35]
[35] Z - D N A A f f i n i t y C h r o m a t o g r a p h y
By RICHARD
FISHEL, PAUL ANZIANO,
and ALEXANDER RICH
Introduction The adsorption of nucleic acids to a column matrix has greatly improved our ability to identify and purify RNA- and DNA-specific metabolic proteins.l,2 In the case of DNA, both duplex and single-stranded as well as specific nucleotide sequences have been added to cellulose, agarose, or Sephacryl matrix materials) ,4 In many of the methods used for linking the DNA to the matrix material, the cross-link is poorly understood, although it generally involves multiple and random sites along the length of the attached substrate DNA. It has been suggested that the efficiency of binding and chromatography of appropriate proteins would increase if the cross-link were specific and situated at the end of the DNA. 5 Recent interest in alternate DNA configurations and their attendant binding proteins has led us to develop a method for quantitative addition of left-handed Z-DNA to a chromatography matrix. 6 The technique utilizes the stable interactions inherent in the avidin-biotin bond as a point of attachment and is similar to previously reported methods. 7,8 We have recently used this material to identify and purify Z-DNA-binding proteins .9 Z-DNA is a left-handed conformation of the DNA double helix. Although both right-handed and left-handed conformations have antiparallel sugar phosphate backbones, they differ considerably in their external form (Fig. 1). Z-DNA has a dinucleotide repeat, and the base pairs are "flipped over" relative to the right-handed B-DNA conformation. Considerable work has been carried out on the interconversion between these 1 H. Shaller, C. Nusslein, F. Bonhoeffer, C. Kurz, and I. Nietzschmann, Eur. J. Biochem. 26, 474 (1972). 2 L. Yarbrough and I. Hurwitz, J. Biol. Chem. 249, 5394 (1974). 3 G. Herrick and B. Alberts, this series, Vol. 21D, p. 198. 4 S. H. Hall and E. A. Smuckler, Biochemistry 13, 3795 (1974). 5 p. T. Gilham, this series, Vol. 21D, p. 191. 6 C. J. Harris, S. P. Moore, and R. Fishel, submitted for publication. 7 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 8 H. Delius, Nucleic Acids Res. 13, 5457 (1985). 9 R. A. Fishel, K. Detmer, and A. Rich, Proc. Natl. Acad. Sci. U.S.A. 85, 36 (1988).
METHODS IN ENZYMOLOGY. VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[35]
D N A COLUMN
329
Maj, Groc Groove Minor Groove
Z-DNA B-DNA FIG. 1. van der Waals diagrams of Z-DNA and B-DNA. Heavy solid lines are drawn from phosphate to phosphate and illustrate the irregularity of the Z-DNA backbone. t w o f o r m s ( r e v i e w e d in R e f s . 10 a n d 1 I). T h e r e is a n e q u i l i b r i u m b e t w e e n B- a n d Z - D N A , b u t Z - D N A is a h i g h e r e n e r g y f o r m t h a t r e q u i r e s s o m e t h i n g to s t a b i l i z e it. T h e D N A p o l y m e r p o l y ( d C - d G ) f o r m s Z - D N A r e a d i l y in 4 M N a C I s o l u t i o n , 12,j3 a n d it c a n b e s t a b i l i z e d in t h e l e f t - h a n d e d f o r m b y b r o m i n a t i o n o f c y t o s i n e at t h e C-5 p o s i t i o n o r b y b r o m i n a t i o n o f g u a n i n e at C-8.14:5 S e v e r a l s u c h c h e m i c a l m o d i f i c a t i o n s h a v e b e e n u s e d to s t a b i l i z e p o l y ( d C - d G ) in t h e Z - D N A c o n f o r m a t i o n in a l o w - s a l t s o l u t i o n . ~0A. Rich, A. Nordheim, and A. H. J. Wang, Annu. Reo. Biochem. 53, 791 (1984). N T. M. Jovin and D. M. Soumpasis, Annu. Rev. Phys. Chem. 38, 521 (1987). 12F. M. Pohl and T. M. Jovin, J. Mol. Biol. 67, 375 (1972). 13T. J. Thamann, R. C. Lord, A. H. J. Wang, and A. Rich, Nucleic Acids Res. 9, 5443 (1981). ~4L. P. Mclntosh, I. Greiger, F. Eckstein, D. A. Zarling, J. H. van de Sande, and T. M. Jovin, Nature (London) 294, 83 (1983). ,5 R. D. Hotchkiss, J. Biol. Chem. 175, 315 (1948).
330
APPLICATIONS
poly(dG-dC) B-DNA
Agor°se -~AA(Avidin StreP~A
HinPI
B-DNA Klenow polymerose
[35]
)
L._ GCl Biotin -
[ Biotin-dCTP dGTP
Z- DNA
B(Biotin) B-DNA
I GC
I
B
4MNoCI Br 2
Z- DNA I
B
B I
I
Biotin Z-DNA
FIG. 2. Flow diagram for the preparation of Z-DNA affinity columns.
In this chapter we describe in detail several methods for preparing Z-DNA that is stable under physiological conditions. This material is then cross-linked to an agarose column matrix at its ends via biotin and streptavidin for use as a chromatography medium. A simple reproducible method for attaching biotin to the ends of virtually any DNA molecule is described. A generalized flow diagram for the construction of a Z-DNA affinity column is shown in Fig. 2.
Reagents Ultrapure Tris-HC1, Tris-OH, sodium chloride, and EDTA are purchased from American Research Products Company (Solon, OH). Tris-buffered solutions are prepared by mixing appropriate volumes of 1 M Tris-HCl and 1 M Tris-OH solutions that result in the desired pH (see Buffer Tables in Sigma Catalog). Sodium citrate is purchased from MaUinckrodt (St. Louis, MO) and a stock solution of 0.2 M is prepared at pH 7.2 with sodium hydroxide (Mallinckrodt). A 10-fold dilution of this stock yields a solution with a final pH of 6.8. Liquid bromine is purchased from Aldrich Chemical Co. (Milwaukee, WI), and a stock solution is prepared by overlaying 25 ml of the bromine with 50 ml glass-distilled water in a sintered glass
[35]
DNA COLUMN
331
container. The water phase is saturated with bromine by repeated inversion followed by settling. Poly(dG-dC), 100 OD units, and poly(dG-mSdC), 25 OD units, from Pharmacia (Piscataway, NJ) are dissolved in 10 m M Tris (pH 7.5), 1 m M EDTA (TE buffer) and biotinylated as described below. The DNA is generally dissolved at a concentration of 100 A280units/ml. A bubbler attachment for a nitrogen gas source is constructed by heat sealing the end of a 10-cm piece of PE240 tubing (Fisher, Pittsburgh, PA) with flamed forceps. The end of the sealed tube is then pincushioned with a 22-gauge needle. Biotinylated-dCTP is purchased from ENZO Biochemicals (New York, NY) as a 0.3 m M solution, dGTP is purchased from Pharmacia Biochemicals and dissolved in 0.1 m M EDTA (pH 8.0). The nucleotide is neutralized to pH 7.0 with 2 M NaOH and the concentration checked spectrophotometrically. HinPI and Escherichia coil DNA polymerase I Klenow fragment are purchased from New England Biolabs (Beverly, MA). Biotinagarose and streptavidin are purchased from Sigma Chemical Co. (St. Louis, MO).
Biotinylation of DNA An enzymatic DNA synthesis using the Klenow fragment ~6of E. coli polymerase I is the basis for the biotinylation reaction. Virtually any DNA substrate can be used as a substrate for biotinylation if it contains a G or an A on the template strand, since both biotinylated dCTP and dUTP are commercially available. To biotinylate poly(dG-dC), the polymer solution with an average length of 4 kb and varying between 0.5 and 25 kb is first partially restricted with HinPI. A 2 mg/ml poly(dG-dC) solution containing 100 units/ml of HinPI is reduced to an average length of 1.5 kb (varying between 0.1 and 9 kb) after incubation for 6 hr at 37 ° under the recommended buffer conditions. This digestion leaves a 2-nucleotide 5' overhang that is a template for fill-in DNA synthesis. Poly(dG-mSdC), however, is resistant to digestion by the three known restriction enzymes that recognize alternating d(GC)n sequences. Thus, an alternate method for generating single-stranded tails that are substrates for DNA synthesis must be devised. A convenient method appears to be sonication, although the energy required to shear DNA must be determined empirically for each instrument. We have found that three short bursts on ice (30 sec, duty cycle 3 at 60%) with a Bronson microtip ~6 H. Jacobsen, H. Klenow, and K. Overgaard-Hansen, Eur. J. Biochem. 45, 623 (1974).
332
APPLICATIONS
[35]
TABLE I EFFICIENCY OF BIOTINYLATION POLYMERASE REACTIONa
Enzyme
Incorporation b (%)
Escherichia coli polymerase I Escherichia coli Klenow fragment Micrococcus luteus polymerase T4 DNA polymerase T7 DNA polymerase Avian myeloblastosis virus (AMV) reverse transcriptase
4.1 8.8 1.3 6.0 0.8 9.2
a The template for polymerase was partially HinPI-restricted poly(dG-dC). b Using [3H]dGTP and biotin-dCTP. The second nucleotide added in this reaction is guanine. The calculated maximum incorporation for polynucleotides of average length 2500 bp is approximately 9%. Reaction conditions were as described by the manufacturer at 37° for 1 hr with 100 U/ml of enzyme and 2 mg/ml of poly(dG-dC) DNA.
sonicator reduces the average length of the polymer to 1-2 kb (varying between 0.1 and 7 kb). Many of these reduced-length fragments appear to be templates for DNA synthesis in the biotinylation reaction as shown below.
DNA Synthesis We have tested several polymerase enzymes for efficiency of biotinylation using biotin-dCTP and [3H]dGTP. The results (Table I) suggest that the Klenow fragment of DNA polymerase I is the most efficient fill-in enzyme for this reaction. We use the following conditions: 40 m M potassium phosphate (pH 7.5), 6.6 m M MgC12, 1.0 m M 2-mercaptoethanol, 0.5 m M dGTP, 6/xM biotin-dCTP, I00 U/ml Klenow fragment, and 20 A260 units/ml DNA. Incubation at 25 ° for 16 hr is sufficient to label over 95% of the DNA molecules on at least one end, based on their ability to be retained on a column matrix. Unincorporated biotinylated nucleotides must be removed from the DNA in order to maximize binding to the column matrix. This is accomplished during the bromination step for the poly(dG-dC) polymer by ultrafiltration. Unincorporated nucleotides can be removed from the poly(dG-msdC) polymer by dialysis, ultrafiltration, or molecular sieve chromatography.
Preparation of Z - D N A The most convenient commercially available substrate for use in the production of large quantities of stable Z-DNA is poly(dG-dC). The con-
D N A COLUMN
[35]
+0.50 I"'i': A O O t.m
'
'
...,
",,,`
C
'
333
'
'
Untreated
APPLICATIONS
[35]
[35] Z - D N A A f f i n i t y C h r o m a t o g r a p h y
By RICHARD
FISHEL, PAUL ANZIANO,
and ALEXANDER RICH
Introduction The adsorption of nucleic acids to a column matrix has greatly improved our ability to identify and purify RNA- and DNA-specific metabolic proteins.l,2 In the case of DNA, both duplex and single-stranded as well as specific nucleotide sequences have been added to cellulose, agarose, or Sephacryl matrix materials) ,4 In many of the methods used for linking the DNA to the matrix material, the cross-link is poorly understood, although it generally involves multiple and random sites along the length of the attached substrate DNA. It has been suggested that the efficiency of binding and chromatography of appropriate proteins would increase if the cross-link were specific and situated at the end of the DNA. 5 Recent interest in alternate DNA configurations and their attendant binding proteins has led us to develop a method for quantitative addition of left-handed Z-DNA to a chromatography matrix. 6 The technique utilizes the stable interactions inherent in the avidin-biotin bond as a point of attachment and is similar to previously reported methods. 7,8 We have recently used this material to identify and purify Z-DNA-binding proteins .9 Z-DNA is a left-handed conformation of the DNA double helix. Although both right-handed and left-handed conformations have antiparallel sugar phosphate backbones, they differ considerably in their external form (Fig. 1). Z-DNA has a dinucleotide repeat, and the base pairs are "flipped over" relative to the right-handed B-DNA conformation. Considerable work has been carried out on the interconversion between these 1 H. Shaller, C. Nusslein, F. Bonhoeffer, C. Kurz, and I. Nietzschmann, Eur. J. Biochem. 26, 474 (1972). 2 L. Yarbrough and I. Hurwitz, J. Biol. Chem. 249, 5394 (1974). 3 G. Herrick and B. Alberts, this series, Vol. 21D, p. 198. 4 S. H. Hall and E. A. Smuckler, Biochemistry 13, 3795 (1974). 5 p. T. Gilham, this series, Vol. 21D, p. 191. 6 C. J. Harris, S. P. Moore, and R. Fishel, submitted for publication. 7 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 8 H. Delius, Nucleic Acids Res. 13, 5457 (1985). 9 R. A. Fishel, K. Detmer, and A. Rich, Proc. Natl. Acad. Sci. U.S.A. 85, 36 (1988).
METHODS IN ENZYMOLOGY. VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[35]
D N A COLUMN
329
Maj, Groc Groove Minor Groove
Z-DNA B-DNA FIG. 1. van der Waals diagrams of Z-DNA and B-DNA. Heavy solid lines are drawn from phosphate to phosphate and illustrate the irregularity of the Z-DNA backbone. t w o f o r m s ( r e v i e w e d in R e f s . 10 a n d 1 I). T h e r e is a n e q u i l i b r i u m b e t w e e n B- a n d Z - D N A , b u t Z - D N A is a h i g h e r e n e r g y f o r m t h a t r e q u i r e s s o m e t h i n g to s t a b i l i z e it. T h e D N A p o l y m e r p o l y ( d C - d G ) f o r m s Z - D N A r e a d i l y in 4 M N a C I s o l u t i o n , 12,j3 a n d it c a n b e s t a b i l i z e d in t h e l e f t - h a n d e d f o r m b y b r o m i n a t i o n o f c y t o s i n e at t h e C-5 p o s i t i o n o r b y b r o m i n a t i o n o f g u a n i n e at C-8.14:5 S e v e r a l s u c h c h e m i c a l m o d i f i c a t i o n s h a v e b e e n u s e d to s t a b i l i z e p o l y ( d C - d G ) in t h e Z - D N A c o n f o r m a t i o n in a l o w - s a l t s o l u t i o n . ~0A. Rich, A. Nordheim, and A. H. J. Wang, Annu. Reo. Biochem. 53, 791 (1984). N T. M. Jovin and D. M. Soumpasis, Annu. Rev. Phys. Chem. 38, 521 (1987). 12F. M. Pohl and T. M. Jovin, J. Mol. Biol. 67, 375 (1972). 13T. J. Thamann, R. C. Lord, A. H. J. Wang, and A. Rich, Nucleic Acids Res. 9, 5443 (1981). ~4L. P. Mclntosh, I. Greiger, F. Eckstein, D. A. Zarling, J. H. van de Sande, and T. M. Jovin, Nature (London) 294, 83 (1983). ,5 R. D. Hotchkiss, J. Biol. Chem. 175, 315 (1948).
330
APPLICATIONS
poly(dG-dC) B-DNA
Agor°se -~AA(Avidin StreP~A
HinPI
B-DNA Klenow polymerose
[35]
)
L._ GCl Biotin -
[ Biotin-dCTP dGTP
Z- DNA
B(Biotin) B-DNA
I GC
I
B
4MNoCI Br 2
Z- DNA I
B
B I
I
Biotin Z-DNA
FIG. 2. Flow diagram for the preparation of Z-DNA affinity columns.
In this chapter we describe in detail several methods for preparing Z-DNA that is stable under physiological conditions. This material is then cross-linked to an agarose column matrix at its ends via biotin and streptavidin for use as a chromatography medium. A simple reproducible method for attaching biotin to the ends of virtually any DNA molecule is described. A generalized flow diagram for the construction of a Z-DNA affinity column is shown in Fig. 2.
Reagents Ultrapure Tris-HC1, Tris-OH, sodium chloride, and EDTA are purchased from American Research Products Company (Solon, OH). Tris-buffered solutions are prepared by mixing appropriate volumes of 1 M Tris-HCl and 1 M Tris-OH solutions that result in the desired pH (see Buffer Tables in Sigma Catalog). Sodium citrate is purchased from MaUinckrodt (St. Louis, MO) and a stock solution of 0.2 M is prepared at pH 7.2 with sodium hydroxide (Mallinckrodt). A 10-fold dilution of this stock yields a solution with a final pH of 6.8. Liquid bromine is purchased from Aldrich Chemical Co. (Milwaukee, WI), and a stock solution is prepared by overlaying 25 ml of the bromine with 50 ml glass-distilled water in a sintered glass
[35]
DNA COLUMN
331
container. The water phase is saturated with bromine by repeated inversion followed by settling. Poly(dG-dC), 100 OD units, and poly(dG-mSdC), 25 OD units, from Pharmacia (Piscataway, NJ) are dissolved in 10 m M Tris (pH 7.5), 1 m M EDTA (TE buffer) and biotinylated as described below. The DNA is generally dissolved at a concentration of 100 A280units/ml. A bubbler attachment for a nitrogen gas source is constructed by heat sealing the end of a 10-cm piece of PE240 tubing (Fisher, Pittsburgh, PA) with flamed forceps. The end of the sealed tube is then pincushioned with a 22-gauge needle. Biotinylated-dCTP is purchased from ENZO Biochemicals (New York, NY) as a 0.3 m M solution, dGTP is purchased from Pharmacia Biochemicals and dissolved in 0.1 m M EDTA (pH 8.0). The nucleotide is neutralized to pH 7.0 with 2 M NaOH and the concentration checked spectrophotometrically. HinPI and Escherichia coil DNA polymerase I Klenow fragment are purchased from New England Biolabs (Beverly, MA). Biotinagarose and streptavidin are purchased from Sigma Chemical Co. (St. Louis, MO).
Biotinylation of DNA An enzymatic DNA synthesis using the Klenow fragment ~6of E. coli polymerase I is the basis for the biotinylation reaction. Virtually any DNA substrate can be used as a substrate for biotinylation if it contains a G or an A on the template strand, since both biotinylated dCTP and dUTP are commercially available. To biotinylate poly(dG-dC), the polymer solution with an average length of 4 kb and varying between 0.5 and 25 kb is first partially restricted with HinPI. A 2 mg/ml poly(dG-dC) solution containing 100 units/ml of HinPI is reduced to an average length of 1.5 kb (varying between 0.1 and 9 kb) after incubation for 6 hr at 37 ° under the recommended buffer conditions. This digestion leaves a 2-nucleotide 5' overhang that is a template for fill-in DNA synthesis. Poly(dG-mSdC), however, is resistant to digestion by the three known restriction enzymes that recognize alternating d(GC)n sequences. Thus, an alternate method for generating single-stranded tails that are substrates for DNA synthesis must be devised. A convenient method appears to be sonication, although the energy required to shear DNA must be determined empirically for each instrument. We have found that three short bursts on ice (30 sec, duty cycle 3 at 60%) with a Bronson microtip ~6 H. Jacobsen, H. Klenow, and K. Overgaard-Hansen, Eur. J. Biochem. 45, 623 (1974).
332
APPLICATIONS
[35]
TABLE I EFFICIENCY OF BIOTINYLATION POLYMERASE REACTIONa
Enzyme
Incorporation b (%)
Escherichia coli polymerase I Escherichia coli Klenow fragment Micrococcus luteus polymerase T4 DNA polymerase T7 DNA polymerase Avian myeloblastosis virus (AMV) reverse transcriptase
4.1 8.8 1.3 6.0 0.8 9.2
a The template for polymerase was partially HinPI-restricted poly(dG-dC). b Using [3H]dGTP and biotin-dCTP. The second nucleotide added in this reaction is guanine. The calculated maximum incorporation for polynucleotides of average length 2500 bp is approximately 9%. Reaction conditions were as described by the manufacturer at 37° for 1 hr with 100 U/ml of enzyme and 2 mg/ml of poly(dG-dC) DNA.
sonicator reduces the average length of the polymer to 1-2 kb (varying between 0.1 and 7 kb). Many of these reduced-length fragments appear to be templates for DNA synthesis in the biotinylation reaction as shown below.
DNA Synthesis We have tested several polymerase enzymes for efficiency of biotinylation using biotin-dCTP and [3H]dGTP. The results (Table I) suggest that the Klenow fragment of DNA polymerase I is the most efficient fill-in enzyme for this reaction. We use the following conditions: 40 m M potassium phosphate (pH 7.5), 6.6 m M MgC12, 1.0 m M 2-mercaptoethanol, 0.5 m M dGTP, 6/xM biotin-dCTP, I00 U/ml Klenow fragment, and 20 A260 units/ml DNA. Incubation at 25 ° for 16 hr is sufficient to label over 95% of the DNA molecules on at least one end, based on their ability to be retained on a column matrix. Unincorporated biotinylated nucleotides must be removed from the DNA in order to maximize binding to the column matrix. This is accomplished during the bromination step for the poly(dG-dC) polymer by ultrafiltration. Unincorporated nucleotides can be removed from the poly(dG-msdC) polymer by dialysis, ultrafiltration, or molecular sieve chromatography.
Preparation of Z - D N A The most convenient commercially available substrate for use in the production of large quantities of stable Z-DNA is poly(dG-dC). The con-
D N A COLUMN
[35]
+0.50 I"'i': A O O t.m
'
'
...,
",,,`
C
'
333
'
'
Untreated
