Proc. Natl. Acad. Sci. USA
Vol. 73, No. 8, pp. 2644-2648, August 1976 Biochemistry
Electron microscopy of DNA crosslinked with trimethylpsoralen: Test of the secondary structure of eukaryotic inverted repeat sequences (cruciforms/electron microscopy under denaturing conditions/glyoxal)
THOMAS R. CECH AND MARY Lou PARDUE Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139
Communicated by Alexander Rich, May 21, 1976
It has been suggested that inverted repeat ABSTRACT (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (mes-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3rpsoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the merpsoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.
strands would accomplish this if (a) the crosslinks were frequent enough to include the inverted repeat sequences, and (b) the crosslinking agent did not disrupt the structure. After such crosslinking, the DNA base pairing would be prevented from undergoing extensive rearrangements via branch migration during DNA purification and electron microscopy. Psoralen and its derivatives intercalate in the DNA helix and undergo a photochemical reaction with pyrinidines of opposite strands, forming covalent crosslinks as well as monoadducts (15-17). The advantages of these Psoralen compounds for DNA crosslinking include their high specificity for DNA compared with proteins, the stability of the interstrand bonds, the high efficiency of the reaction, and the lack of the DNA degradation that accompanies crosslinking with other reagents (17-19). Furthermore, Psoralen can be used to crosslink DNA inside of living cells (15-18). Psoralen crosslinking therefore seemed well suited to a study of the secondary structure of inverted repeat sequences in intact cells. Mouse tissue culture cells were chosen for this study because the size and frequency of the inverted repeat sequences from this source are known in detail (3, 8). The study concerns only inverted repeats with sequences longer than 200 nucleotides (NT), the size that can be consistently identified by electron microscopy (8). A homogeneous class of inverted repeat sequences, the macronuclear ribosomal DNA (rDNA) of Tetrahymena (11), was used for in vitro tests of the crosslinking of cruciforms.
A small percentage of the nuclear DNA of eukaryotes is organized in the form of inverted repeat sequences, also called palindromes (1-10). After the DNA is denatured, the inverted repeat sequences undergo rapid, intramolecular renaturation and are seen in the electron microscope as hairpin structures containing hundreds or thousands of base pairs (4, 5, 7, 8). In addition to the size of these sequences, we know their distribution in genomes, their sequence complexity, and the fidelity with which the sequences are repeated. These facts, however, have revealed very little about their origin or function. The sequence symmetry of inverted repeats makes possible two base-paired configurations, a linear form and a "cruciform" (30, 31; Fig. 1). These alternate secondary structures of the same palindromic sequence have recently been observed in vitro (11). If cruciforms occurred in vivo, they-might function as protein recognition sites (12) or sites for genetic recombination (13), or might provide a method for conserving the complementarity of the two halves of inverted repeat sequences (4). Wilson and Thomas (4) saw no cruciforms in purified DNA, but suggested that cruciforms might be stabilized by chromosomal proteins in vivo. In purified DNA the linear form should be the thermodynamically favored form (14). In order to observe in purified DNA the configuration that inverted repeat sequences have in vivo, be it linear or cruciform, the DNA configuration must be "fixed" while it is inside the undisrupted cell. Covalent crosslinking of the two DNA
MATERIALS AND METHODS Crosslinking DNA. SVT2 (simian virus 40-transformed) mouse tissue culture cells were supplied by Theodore and Tucker Gurney. They were grown as monolayers in 25 cm2 plastic tissue culture flasks (Falcon) and labeled with [methyl-3H]thymidine as described previously (3). When the cells were in the log phase of growth (1.2 X 105/cm2), the medium in each flask was replaced by 3 ml of sterile phosphatebuffered saline (with MgCl2 and CaCl2) saturated with 4,5',8-trimethylpsoralen (mes-psoralen) (final concentration 3 ,gg/ml). The me3-psoralen, a gift of the Paul B. Elder Drug Co. (Brian, Ohio), was dissolved in 50% ethanol at 60 ,gg/ml. Control cultures received the same buffer with 50% ethanol replacing the me3-psoralen solution. The flasks were irradiated from the bottom with 365 nm light by means of a General Electric F15T8 BLB fluorescent lamp. The lamp was 2.3 cm away from the cells, with a one-quarter inch (6 mm) thickness of Plexiglas filtering out essentially all light of wavelengths below 340 nm (A340 > 3). The incident light intensity was 0.72 mW/cm2, determined by potassium ferrioxalate actinometry (20). The buffer in the flasks was at a constant temperature of 28° during the irradiation. Light microscopy showed that most
Abbreviations: me3-psoralen, 4,5',S-trimethylpsoralen; NT, nucleotide or nucleotide pair; kb, kilobase, 1000 NT; rDNA, DNA sequences coding for 17S and 25S ribosomal RNA with adjacent spacer se-
quences.
2644
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Cech and Pardue C
.
b' a' +F a b c x c' b' a' : -_l - Cba cmx v -F
-F
.
rosslink
FIG. 1. The cruciform hypothesis. A segment of a long DNA molecule contains an inverted repeat sequence (the sequence abc followed by the complementary sequence a'b'c' in reverse order). A sequence which is not self-complementary (x) may separate the two parts of the inverted repeat sequence. Two possible alternate configurations of the molecule are shown. Factors (F) present inside the cell may stabilize the cruciform relative to the linear structure. Upon the removal of these factors, the cruciform DNA could rezipper into the linear form by double-stranded branch migration. Crosslinking the opposite strands of the DNA double helix should stabilize the structure and permit cruciforms to be seen even in purified DNA, when the stabilizing factors have been removed. The thin lines represent DNA base pairs, the thick lines are covalent crosslinks.
of the cells remained attached to the plastic during the me3psoralen plus irradiation treatment. Purified DNA was crosslinked in a manner similar to that described above. Drops of DNA solution (10 gg/ml) containing 3 ,ug/ml of me3-psoralen were irradiated in a humidified plastic petri dish. Isolation of Crosslinked Mouse DNA. After crosslinking, the SVT2 cells were removed from the flasks with trypsin and harvested by centrifugation. DNA was extracted as described previously (3), with the addition of a phenol extraction before the first chloroform extraction. All steps prior to the phenol extraction were done in low room light. Isolation of Tetrahymena rDNA. Tetrahymena pyrtformis strain B VII, provided by Kathleen Karrer, was grown in sterile 1% proteose peptone. DNA was extracted by the same method used for the mouse cells, except that two ribonuclease treatments were performed. Each was followed by Pronase treatment and chloroform extractions. The rDNA was isolated by two cycles of Hg++-Cs2SO4 density gradient centrifugation (11). About 98% by weight of this DNA was unit length (5.9 Jim) molecules when spread from 50% formamide. All molecules were linear except for rare replicating forms. Electron Microscopy. DNA was spread from 50% formamide according to the isodenaturing technique of Davis et al. (21). Grids were stained with uranyl acetate and shadowed with 80% Pt-20% Pd. A Jeolco JEM1OOB electron microscope was used. Plates were exposed at a magnification of X15,000. In experiments designed to completely denature DNA in order to see crosslinks, the DNA was treated with glyoxal in the following manner. Ten microliters of DNA (dialyzed against 0.10 M Tris.HCI buffer, 0.01 M EDTA, pH 8.4) was mixed with 73 ,ul of 99% formamide (Matheson, Colman, and Bell), 1O0ul of 0.10 M NaH2PO4-Na2HPO4 buffer, 0.01 M EDTA, pH 6.9, and 7 gl of glyoxal (Eastman Technical Grade, 40% in H20). The final glyoxal concentration was 0.5 M. The solution was heated at 370 for 45-60 min. The combination of low ionic strength and high formamide concentration results in complete denaturation of DNA at 370. The glyoxal reaction (22) prevents base pairing (predominantly G.C base pairing) even when the DNA is returned to more stabile conditions (23, 24). After the denaturation, 10 Al of the above solution was diluted into 43
2645
of formamide, 10.ul of buffer (1.0 M Tris buffer, 0.1 M EDTA, pH 8.4), and 37 1l of H20 and spread by the standard 50% formamide method. Single-stranded, circular phage 4X174 DNA, provided by Claire Moore, was added to some of the samples before glyoxal treatment as a molecular weight standard [5.25 kilobases (kb)]. This glyoxal technique is a variation of the method developed by Hanson et al. (28) for observing me3-psoralen crosslinks. They employed formaldehyde instead of glyoxal to prevent base pairing. We have obtained equivalent results with the two reagents, but prefer the glyoxal technique because it gives more consistently usable preparations. RESULTS The distribution of crosslinks in DNA reacted with mex psoralen inside growing cells Mouse tissue culture cells were treated with me3-psoralen and/or irradiation as summarized in Table 1. A portion of the DNA extracted from each group of cells was examined by electron microscopy under totally denaturing conditions to determine the number and distribution of crosslinks (28). After the denaturation treatment, sample SV6 showed alternating single-stranded bubbles and apparently double-stranded regions (Fig. 2a and b). The regions that appeared double-stranded are interpreted to consist of DNA that is densely crosslinked, such that the two denatured strands are constrained to follow each other closely (Fig. 2e). The sizes of bubbles and double-strand regions changed as expected with increasing irradiation dose (Fig. 2c and d and Table 1). When the control samples (SV1-3) were denatured and spread for microscopy by the same method, only single-stranded DNA was seen. The electron microscopic estimates of crosslinking density were substantiated by denaturation-renaturation experiments. Covalent crosslinks hold the two strands of the double helix in register during denaturation, allowing rapid, unimolecular "snap-back" renaturation (25). Portions of samples SV1-6 were sonicated to a weight-average molecular weight of 500 NT and then analyzed by spectrophotometric melting-cooling curves and hydroxyapatite chromatography experiments (data not shown). DNA that had not been exposed to crosslinking conditions (samples SV1-3) exhibited only 3% snap-back renaturation, the amount expected from inverted repeat sequences (3, 4). The 500 NT DNA fragments from samples SV5 and SV6, Table 1. Electron microscopic assay of crosslinking
Sample
me3psoralen (3 gg/ml)
Irradiation (min)
Average distance between crosslinks (NT)
-
* * * *
SV1 SV2 SV3 SV4 SV5
+
0 100 0 1 12
SV6
+
100
+ +
890 (95%)t
Vol. 73, No. 8, pp. 2644-2648, August 1976 Biochemistry
Electron microscopy of DNA crosslinked with trimethylpsoralen: Test of the secondary structure of eukaryotic inverted repeat sequences (cruciforms/electron microscopy under denaturing conditions/glyoxal)
THOMAS R. CECH AND MARY Lou PARDUE Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139
Communicated by Alexander Rich, May 21, 1976
It has been suggested that inverted repeat ABSTRACT (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (mes-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3rpsoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the merpsoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.
strands would accomplish this if (a) the crosslinks were frequent enough to include the inverted repeat sequences, and (b) the crosslinking agent did not disrupt the structure. After such crosslinking, the DNA base pairing would be prevented from undergoing extensive rearrangements via branch migration during DNA purification and electron microscopy. Psoralen and its derivatives intercalate in the DNA helix and undergo a photochemical reaction with pyrinidines of opposite strands, forming covalent crosslinks as well as monoadducts (15-17). The advantages of these Psoralen compounds for DNA crosslinking include their high specificity for DNA compared with proteins, the stability of the interstrand bonds, the high efficiency of the reaction, and the lack of the DNA degradation that accompanies crosslinking with other reagents (17-19). Furthermore, Psoralen can be used to crosslink DNA inside of living cells (15-18). Psoralen crosslinking therefore seemed well suited to a study of the secondary structure of inverted repeat sequences in intact cells. Mouse tissue culture cells were chosen for this study because the size and frequency of the inverted repeat sequences from this source are known in detail (3, 8). The study concerns only inverted repeats with sequences longer than 200 nucleotides (NT), the size that can be consistently identified by electron microscopy (8). A homogeneous class of inverted repeat sequences, the macronuclear ribosomal DNA (rDNA) of Tetrahymena (11), was used for in vitro tests of the crosslinking of cruciforms.
A small percentage of the nuclear DNA of eukaryotes is organized in the form of inverted repeat sequences, also called palindromes (1-10). After the DNA is denatured, the inverted repeat sequences undergo rapid, intramolecular renaturation and are seen in the electron microscope as hairpin structures containing hundreds or thousands of base pairs (4, 5, 7, 8). In addition to the size of these sequences, we know their distribution in genomes, their sequence complexity, and the fidelity with which the sequences are repeated. These facts, however, have revealed very little about their origin or function. The sequence symmetry of inverted repeats makes possible two base-paired configurations, a linear form and a "cruciform" (30, 31; Fig. 1). These alternate secondary structures of the same palindromic sequence have recently been observed in vitro (11). If cruciforms occurred in vivo, they-might function as protein recognition sites (12) or sites for genetic recombination (13), or might provide a method for conserving the complementarity of the two halves of inverted repeat sequences (4). Wilson and Thomas (4) saw no cruciforms in purified DNA, but suggested that cruciforms might be stabilized by chromosomal proteins in vivo. In purified DNA the linear form should be the thermodynamically favored form (14). In order to observe in purified DNA the configuration that inverted repeat sequences have in vivo, be it linear or cruciform, the DNA configuration must be "fixed" while it is inside the undisrupted cell. Covalent crosslinking of the two DNA
MATERIALS AND METHODS Crosslinking DNA. SVT2 (simian virus 40-transformed) mouse tissue culture cells were supplied by Theodore and Tucker Gurney. They were grown as monolayers in 25 cm2 plastic tissue culture flasks (Falcon) and labeled with [methyl-3H]thymidine as described previously (3). When the cells were in the log phase of growth (1.2 X 105/cm2), the medium in each flask was replaced by 3 ml of sterile phosphatebuffered saline (with MgCl2 and CaCl2) saturated with 4,5',8-trimethylpsoralen (mes-psoralen) (final concentration 3 ,gg/ml). The me3-psoralen, a gift of the Paul B. Elder Drug Co. (Brian, Ohio), was dissolved in 50% ethanol at 60 ,gg/ml. Control cultures received the same buffer with 50% ethanol replacing the me3-psoralen solution. The flasks were irradiated from the bottom with 365 nm light by means of a General Electric F15T8 BLB fluorescent lamp. The lamp was 2.3 cm away from the cells, with a one-quarter inch (6 mm) thickness of Plexiglas filtering out essentially all light of wavelengths below 340 nm (A340 > 3). The incident light intensity was 0.72 mW/cm2, determined by potassium ferrioxalate actinometry (20). The buffer in the flasks was at a constant temperature of 28° during the irradiation. Light microscopy showed that most
Abbreviations: me3-psoralen, 4,5',S-trimethylpsoralen; NT, nucleotide or nucleotide pair; kb, kilobase, 1000 NT; rDNA, DNA sequences coding for 17S and 25S ribosomal RNA with adjacent spacer se-
quences.
2644
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Cech and Pardue C
.
b' a' +F a b c x c' b' a' : -_l - Cba cmx v -F
-F
.
rosslink
FIG. 1. The cruciform hypothesis. A segment of a long DNA molecule contains an inverted repeat sequence (the sequence abc followed by the complementary sequence a'b'c' in reverse order). A sequence which is not self-complementary (x) may separate the two parts of the inverted repeat sequence. Two possible alternate configurations of the molecule are shown. Factors (F) present inside the cell may stabilize the cruciform relative to the linear structure. Upon the removal of these factors, the cruciform DNA could rezipper into the linear form by double-stranded branch migration. Crosslinking the opposite strands of the DNA double helix should stabilize the structure and permit cruciforms to be seen even in purified DNA, when the stabilizing factors have been removed. The thin lines represent DNA base pairs, the thick lines are covalent crosslinks.
of the cells remained attached to the plastic during the me3psoralen plus irradiation treatment. Purified DNA was crosslinked in a manner similar to that described above. Drops of DNA solution (10 gg/ml) containing 3 ,ug/ml of me3-psoralen were irradiated in a humidified plastic petri dish. Isolation of Crosslinked Mouse DNA. After crosslinking, the SVT2 cells were removed from the flasks with trypsin and harvested by centrifugation. DNA was extracted as described previously (3), with the addition of a phenol extraction before the first chloroform extraction. All steps prior to the phenol extraction were done in low room light. Isolation of Tetrahymena rDNA. Tetrahymena pyrtformis strain B VII, provided by Kathleen Karrer, was grown in sterile 1% proteose peptone. DNA was extracted by the same method used for the mouse cells, except that two ribonuclease treatments were performed. Each was followed by Pronase treatment and chloroform extractions. The rDNA was isolated by two cycles of Hg++-Cs2SO4 density gradient centrifugation (11). About 98% by weight of this DNA was unit length (5.9 Jim) molecules when spread from 50% formamide. All molecules were linear except for rare replicating forms. Electron Microscopy. DNA was spread from 50% formamide according to the isodenaturing technique of Davis et al. (21). Grids were stained with uranyl acetate and shadowed with 80% Pt-20% Pd. A Jeolco JEM1OOB electron microscope was used. Plates were exposed at a magnification of X15,000. In experiments designed to completely denature DNA in order to see crosslinks, the DNA was treated with glyoxal in the following manner. Ten microliters of DNA (dialyzed against 0.10 M Tris.HCI buffer, 0.01 M EDTA, pH 8.4) was mixed with 73 ,ul of 99% formamide (Matheson, Colman, and Bell), 1O0ul of 0.10 M NaH2PO4-Na2HPO4 buffer, 0.01 M EDTA, pH 6.9, and 7 gl of glyoxal (Eastman Technical Grade, 40% in H20). The final glyoxal concentration was 0.5 M. The solution was heated at 370 for 45-60 min. The combination of low ionic strength and high formamide concentration results in complete denaturation of DNA at 370. The glyoxal reaction (22) prevents base pairing (predominantly G.C base pairing) even when the DNA is returned to more stabile conditions (23, 24). After the denaturation, 10 Al of the above solution was diluted into 43
2645
of formamide, 10.ul of buffer (1.0 M Tris buffer, 0.1 M EDTA, pH 8.4), and 37 1l of H20 and spread by the standard 50% formamide method. Single-stranded, circular phage 4X174 DNA, provided by Claire Moore, was added to some of the samples before glyoxal treatment as a molecular weight standard [5.25 kilobases (kb)]. This glyoxal technique is a variation of the method developed by Hanson et al. (28) for observing me3-psoralen crosslinks. They employed formaldehyde instead of glyoxal to prevent base pairing. We have obtained equivalent results with the two reagents, but prefer the glyoxal technique because it gives more consistently usable preparations. RESULTS The distribution of crosslinks in DNA reacted with mex psoralen inside growing cells Mouse tissue culture cells were treated with me3-psoralen and/or irradiation as summarized in Table 1. A portion of the DNA extracted from each group of cells was examined by electron microscopy under totally denaturing conditions to determine the number and distribution of crosslinks (28). After the denaturation treatment, sample SV6 showed alternating single-stranded bubbles and apparently double-stranded regions (Fig. 2a and b). The regions that appeared double-stranded are interpreted to consist of DNA that is densely crosslinked, such that the two denatured strands are constrained to follow each other closely (Fig. 2e). The sizes of bubbles and double-strand regions changed as expected with increasing irradiation dose (Fig. 2c and d and Table 1). When the control samples (SV1-3) were denatured and spread for microscopy by the same method, only single-stranded DNA was seen. The electron microscopic estimates of crosslinking density were substantiated by denaturation-renaturation experiments. Covalent crosslinks hold the two strands of the double helix in register during denaturation, allowing rapid, unimolecular "snap-back" renaturation (25). Portions of samples SV1-6 were sonicated to a weight-average molecular weight of 500 NT and then analyzed by spectrophotometric melting-cooling curves and hydroxyapatite chromatography experiments (data not shown). DNA that had not been exposed to crosslinking conditions (samples SV1-3) exhibited only 3% snap-back renaturation, the amount expected from inverted repeat sequences (3, 4). The 500 NT DNA fragments from samples SV5 and SV6, Table 1. Electron microscopic assay of crosslinking
Sample
me3psoralen (3 gg/ml)
Irradiation (min)
Average distance between crosslinks (NT)
-
* * * *
SV1 SV2 SV3 SV4 SV5
+
0 100 0 1 12
SV6
+
100
+ +
890 (95%)t
