(Received for publication, September 11, 1996, and in revised form, November 5, 1996)
From the Laboratory of Molecular Genetics, the § Laboratory of Molecular Biophysics, and the Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021
We have developed a novel photocross-linking
technique using free 8-methoxypsoralen and DNA furan-side monoadducts
plus long wave ultraviolet light (UVA). Both sequence-specific (Max)
and nonspecific (RecA and T7 RNA polymerase) DNA-binding proteins were
cross-linked. The macroscopic equilibrium binding constant (~109 M1) and DNase I
footprinting indicated that binding of Max to its cognate sequence
(E-box) was unimpaired by 8-methoxypsoralen and that cross-linking
occurred in normal complexes. RecA protein and T7 RNA polymerase were
cross-linked to a 12-mer DNA furan-side monoadduct with UVA. Cross-link
yields were directly proportional to the UVA dose. Cross-links were
stable to 8 M urea, 1-10% SDS, commonly used alcohols,
and mild acids (5% trichloroacetic acid). The DNA in cross-links was
reversed with 254 nm UV (photoreversal) or with hot base
(base-catalyzed reversal), consistent with (2 + 2) cycloaddition via
the 4
,5
-furan of the psoralen. Comparative action spectra for DNA-DNA
cross-linking and DNA-protein cross-linking revealed that the latter
occurred maximally at 300 nm, while the former occurred maximally at
320 nm. This 20-nm blue shift suggested a higher potential energy
surface for an excited psoralen participating in protein-DNA
cross-linking as compared with DNA-DNA cross-linking. As with DNA-DNA
cross-linking, DNA-protein cross-linking is a two-photon process.
Absorption of the first photon formed a 4
,5
-adduct with DNA, which
then absorbed a second photon, leading to cross-linking to protein.
Based on the action spectra and the known excited states of psoralen,
it is suggested that the triplet n,
* transition localized in the C-2=O of psoralen may be involved in protein-psoralen photoreactions.
The control of gene expression is achieved principally through the
regulatory activities of DNA-binding proteins. Modern methods of x-ray
crystallography and NMR have revealed the nature of the interactions
that govern specificity in DNA-protein recognition. Techniques of
protein-DNA cross-linking have been used either to confirm high
resolution structures in solution or to obtain structural information
where no high resolution structures are available (e.g. see
Refs. 1-5). A number of photoreactive agents such as carbenes,
nitrenes, thio derivatives, benzophenones, halogenated pyrimidines, or
simply 254-nm UV, are routinely employed to achieve protein-DNA
photocross-links (reviewed in Refs. 6-10). Psoralens (Fig. 1) possess
some advantages. Psoralens and their adducts are highly photostable in
ambient room light and stable for months when frozen, and as such are
easy to use. Psoralens can be site-specifically attached to DNA at
pyrimidines (preferentially at 5-TpA sites), and their absorption
bands extend well beyond (up to 410 nm) those of either protein or DNA,
enabling cross-linking without damage to either protein or DNA. In an
ongoing effort to develop photocross-linking methods and to understand
protein-DNA recognition in various systems, we focused on psoralen as a
probe for DNA-protein interactions. Psoralens are potentially useful
because they can react with both DNA and proteins (11-14). Psoralens
(Fig. 1) are furocoumarins used in the treatment of vitiligo, psoriasis
mycoses fungoides, and several other skin diseases (see Refs. 15-17)
for reviews). Upon exposure to long wavelength UV light (UVA; 310-400
nm),1 psoralens react with DNA or RNA to
form covalent adducts (12, 17, 18). In DNA, psoralens react primarily
with thymidine and to a lesser extent with cytosine. Psoralens
photoalkylate DNA by (2 + 2) cycloaddition to the 5,6-double bond of
pyrimidines. Monoaddition occurs via either the 4
,5
-double bond of
the furan (Fig. 1, III) or the 3,4-double bond of pyrone of
psoralen (see Fig. 1 of Ref. 19 for the molecular structures of
psoralen adducts in DNA). Absorption of a second photon by furan-side
monoadducts (Fig. 1, III), after a 1-µs relaxation,
results in the cross-linking of the furan-side adduct to the thymine on
the complementary strand (20). Recently, NMR-derived structures of the
DNA furan side and the interstrand cross-link have been solved
(19).
The covalent photobinding of psoralens to proteins was documented after
the discovery of their reactivity with DNA (21-25). Compared with
their DNA photochemistry, the reactivity of psoralens with proteins is
poorly understood. Psoralen derivatives induce protein-DNA cross-links
in vivo (26-28) and in vitro (13). The structural basis of the chemical adducts among psoralen, proteins, and
amino acids is unknown. Psoralens were shown to photochemically inactivate a number of enzymes. Some examples are lysozyme, glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, ribonuclease, nuclear
histones, and DNA polymerases (see Ref. 11 for review). Many different
amino acids in the proteins were shown to react with free psoralens
including Trp and Tyr (11, 28). Singlet oxygen
(1g;1O*2) was
implicated in the formation of the photoadducts between proteins and
psoralen (29-33). Photoreactive mechanisms not involving 1O*2 have been proposed (see Ref. 11 and
references therein). We have recently isolated and chemically
characterized a psoralen-Tyr photoadduct.2
One of us previously reported the development of a technique for photocross-linking DNA-binding proteins to DNA via psoralen (13, 14). Using this technique, it was shown that promoter DNA binds in the cleft of T7 RNAP, and a nonspecific ssDNA binding site occurred in the fingers domain. The DNA template in an elongation complex was cross-linked to T7 RNAP by placing a site-specific psoralen furan-side monoadduct in the path of an elongating T7 RNAP (13, 35, 36).
Here, we further demonstrate the versatility of the psoralen cross-linking method to study both sequence-specific and nonspecific DNA recognition. As a first step toward an understanding of the different photochemistries of psoralen in DNA-protein cross-linking and DNA-DNA cross-linking, we compared relative action spectra for cross-linking.
Materials
DNAs and ProteinsUnmodified DNAs and site-specific
psoralen furan-side DNA monoadducts were synthesized and purified as
described previously (37). Max-binding 16-mer oligomers were
synthesized and purified locally. Equimolar (1-2 µM)
amounts of complementary oligomers were mixed in 50 mM
Tris-HCl (pH 7.5.), 1 mM MgCl2 and annealed by
heating at 65 °C for 5 min and slowly cooling overnight to room
temperature (~27 °C). The concentrations of DNAs were calculated from their respective molar extinction coefficients at 260 nm (~104 M1 cm
1 per
nucleotide). A subfragment of human Max (amino acids 22-113) was
purified as described previously (38). T7 RNA polymerase was prepared
locally according to published procedures (39). RecA protein was a kind
gift of Dr. Wendy Bedale (Dr. Michael Cox's laboratory at the
University of Wisconsin, Madison, WI). The concentrations of the
purified proteins were estimated using their published extinction
coefficients at 280 nm (
280; in
M
1 cm
1): Max = 2.6 × 103; T7 RNAP = 1.4 × 105; and
RecA = 2.1 × 104. 8-MOP (Fig. 1, I)
and HMT (Fig. 1, II) were purchased from ICN pharmaceuticals (Irvine, CA) and HRI Associates Inc. (Concord, CA),
respectively, and used as described previously (37).
Methods
Irradiation of DNA-Protein Complexes with Continuous Wave UVA LightThe light source was a 200-W mercury/xenon arc lamp (Oriel Corp., Stratford, CT). The lamp housing was fitted with a liquid filter to eliminate IR and a dichroic mirror with >90% reflectance in the 350-450 nm range (Oriel Corp. model 66218). For analytical cross-link preparations, the lamp housing was fitted with a mercury line band pass filter centered at 365-nm with a 10-nm bandwidth (Oriel Corp. model 56531). A 2.8-cm fused silica lens was used to focus the UV light on to the slit of a thermostated 100-µl quartz cuvette (1-cm path length) that contained the reaction mixture. The cuvette was held in a custom-designed cuvette holder thermostated by a circulating Lauda bath containing a 50:50 mixture of water/ethylene glycol. The UVA light impinging and exiting the slit window of the cuvette was measured using an international light meter (model IL1700) fitted with a calibrated UVA probe (model SFD038).
Max-DNA Cross-linking8-MOP was added to 32P-labeled duplex DNA at a molar ratio of 1000:1 and allowed to incubate in the dark at room temperature for 30 min in K+ phosphate-Mg2+ buffer (50 mM K+ phosphate (pH 6.0), 1 mM Mg(OAc)2) or Tris-Mg2+ buffer (10 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM MgCl2, 5 mM dithiothreitol, 10% glycerol). Both buffers gave similar results. Max was then added to the "dark" reaction at a Max monomer:DNA molar ratio of 4:1 or 12:1. The mixture (25 µl) was incubated for 30 min at room temperature (~27 °C) and then irradiated for 3 min with the mercury/xenon arc source (see above) fitted with a dichroic mirror. To 10 µl of the photoreaction, an equal volume of 2% SDS sample buffer was added and heated for 5-10 min at 95 °C. The denatured samples were electrophoresed on a 20% acrylamide, SDS, Tris, Tricine gel (29:1 acrylamide:bisacrylamide). The gel was run overnight at 2 mA/cm. The wet gel was exposed to an x-ray film and subsequently to a phosphor screen.
Noncovalent Binding of Max to Cognate DNAGel mobility shift assays were performed as follows. One-half pmol of 32P-labeled duplex DNA was incubated in K+ phosphate-Mg2+ buffer or Tris-Mg2+ buffer with various amounts of 8-MOP for 10 min in the dark at room temperature (~25 °C). Various amounts of Max (see figure legends) were added, and the incubation was continued for 30 min (25-µl final volume). The reaction was mixed with 5 µl of 50% glycerol and loaded on an 8% acrylamide nondenaturing gel (19:1 acrylamide:bisacrylamide). Before loading the samples, the gel was prerun for 1 h, and the tank buffer was replaced with fresh buffer. The gel (12 cm length × 16 cm width) was run at 10 V/cm until the bromphenol blue dye reached 6 cm from the bottom of the wells. The gels were soaked for 20 min in 5% methanol, 5% acetic acid, 3% glycerol with agitation and then dried under vacuum at 80 °C. The radiolabeled bands were visualized by autoradiography with x-ray film, and subsequently with a phosphor screen.
DNase I Footprinting of Max-DNA ComplexesOne-half pmol of 32P-labeled duplex DNA was incubated in potassium phosphate buffer with various amounts of 8-MOP for 10 min in the dark at room temperature (~25 °C). Various amounts of Max were added, and incubation was continued for 30 min (25-µl final volume). DNase I was added to a final concentration of 140 ng/ml, and incubation was continued for 5 min. The reaction was stopped by adding EDTA to 20 mM. The reaction was then phenol-extracted and ethanol-precipitated, and the DNA was resuspended in TBE (180 mM Tris borate, 2 mM EDTA buffer), 8 M urea dyes and run on a 24% acrylamide denaturing gel.
T7 RNAP or RecA-ssDNA Cross-linking32P-Labeled
12-Maf (5-GAAGCTACGAGC-3
with a psoralen furan-side
monoadduct at dT) was incubated in buffer containing either
50 mM Tris-HCl (pH 8.0), 10 mM
MgCl2, 1 mM dithiothreitol, 0.5 mM
spermidine, 5% glycerol (for T7 RNAP) or 30 mM HEPES, pH 7.8, 10 mM Mg(OAc)2, 150 mM
K+ glutamate, 0.25 mM EDTA, Tween 0.05%, 1 mM dithiothreitol, 0.1 mg/ml acetylated bovine serum
albumin, 1 mM ATP
S (for RecA). The proteins were added
to the DNA and incubated at room temperature for 10 min. The reaction
mixtures (25 µl) were irradiated with mercury/xenon arc source for
various lengths of time (see Fig. 7). The samples were denatured and
processed for SDS gel electrophoresis as described for Max (see above).
The cross-linked samples were run on 8% acrylamide minigels (Bio-Rad
Mini Protean II apparatus, Hercules, CA). The gels were dried and
autoradiographed.
Quantitation of Cross-linking
Cross-link yields are
expressed as arbitrary units of relative cross-linking, defined as
(XLb/(XLb + XLf)) (C/(C + Cf)) × 1000, where XLb is the
integrated band area representing the cross-linked DNA,
XLf is the band area representing the free DNA in
the same lane, C is the band area corresponding to the
position of cross-links in the control (no UVA), and
Cf is the band area corresponding to the free DNA in
the same lane. All band areas were obtained first by deducting
nonspecific background counts in a portion of the gel where no samples
were loaded. Autoradiography was performed with a phosphor screen.
Quantitation of gel bands was carried out on phosphor images of gels
with the aid of the ImageQuant program using a PhosphorImager
(Molecular Dynamics, Mountain View, CA).
To estimate the cross-link yield in terms of the amounts of protein, we
translated the sum total of storage phosphor counts of all cross-linked
bands to the amount of 32P using a standard curve. We
constructed the standard curve using graded amounts of known
[-32P]ATP of known specific radioactivity. Many rows
of [
-32P]ATP were spotted on a 3MM filter paper,
dried, and exposed to a phosphor screen. The same samples were also
counted for 32P in a scintillation counter. The
32P Cerenkov counts of these samples were plotted against
the corresponding storage phosphor counts. The relationship between
32P Cerenkov counts (or the actual amount in pmol of
[32P]ATP) and the corresponding storage phosphor counts
was perfectly linear over a large dynamic range of radioactivity. Using
the standard curve and the observed storage phosphor counts, we
obtained the 32P counts for each cross-linked and free DNA
band. From the specific radioactivity of the 32P label we
calculated the amount of DNA in cross-linked bands. Assuming that one
protein monomer cross-linked per DNA, we calculated the amount of
cross-linked protein. The percentage of protein cross-link yield was
obtained by dividing the pmol of cross-linked protein by pmol of the
total amount of protein in the reaction mixture and multiplying the
result with 100.
All spectra were obtained in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA at room temperature with a Beckman DU60 scanning UV-visible spectrophotometer using a 100-µl masked quartz cuvette of 1-cm path.
Action SpectraMany stock reaction mixtures (each 200 µl)
were prepared at convenient intervals of time and kept on ice during
the course of these experiments. For RecA-DNA cross-linking, each stock
reaction contained 0.5 pmol of 32P-12-mer DNA or
32P-12-Maf DNA, 7 pmol of RecA, 1 mM ATPS in
potassium glutamate buffer (see above for RecA-ssDNA cross-linking
buffer). Stock solutions (200 µl) for 32P-12-mer
DNA/8-mer DNA cross-linking contained 0.5 pmol of
32P-12-mer (or 32P-12-Maf)-8-mer duplex in the
same buffer as for RecA cross-linking. When needed, HMT was added to 3 µg/ml. Without added HMT, the absorbance of the solutions at 300 nm
was negligible. With HMT, it was below 0.2 between 280 and 300 nm. This
minimized inner shielding effects and light scattering. Fifty-µl
aliquots from stock reactions were irradiated at 30 °C for 30 min at
each wavelength. To maintained a relatively uniform absorption
cross-section, the solution was periodically mixed quickly during
irradiation using a micropipette. A Bausch and Lomb monochromator
(model 33-86-01, serial number 2324BK, with a grating of 2700 grooves/mm and 3.2-nm bandwidth at each wavelength) was attached
immediately after the IR filter of the 200-W mercury/xenon arc housing
(see above). No further attachments were made. Monochromator entrance
and exit slits were set at fully open positions, and a light spot of
~2-3 mm was focused on the entrance slit of the quartz cuvette. A
thermopile probe (Scientech, Boulder, CO, model 380101, serial number
4579) coupled to a microvoltmeter (Keittley Instruments) was positioned in line behind the cuvette exit slit, and the light energy was measured
at 5-min intervals during sample irradiation. The sensitivity of the
thermopile probe was independent of wavelength. The light energies at
each wavelength were measured with and without samples in the cuvette.
The following were the measured absolute energy distributions (
= W/cm2): 280 nm, 0.02; 290 nm, 0.04; 300 nm, 0.05; 310 nm,
0.13; 320 nm, 0.1; 330 nm, 0.02; 340 nm, 0.02; 350 nm, 0.01; 360, 0.06; 370 nm, 0.11; 380 nm, 0.04; 390 nm, 0.01. The sample geometry and the
photon dose at each wavelength were the same for each sample. After the
irradiation, the samples were denatured by heating at 95 °C for 5 min and concentrated by heating in a speed vac (Savant) and then run on
10% acrylamide-SDS gels to visualize protein-DNA cross-links or 24%
acrylamide-8 M urea gels to visualize the
[32P]DNA cross-links (13, 37). Quantitation of the gel
bands was carried out by PhosphorImager analysis. Action spectra were normalized for light intensities. The sample composition for each type
of cross-linking experiment, photon density per sample and sample
geometry and other conditions during photoreactions were the same.
Action spectra are expressed in arbitrary units as relative cross-linking (defined above) of either DNA to DNA or DNA to
protein.
Max belongs to a class of sequence-specific DNA-binding
eucaryotic transcription factors that contain a
basic/helix-loop-helix/zipper motif (38, 40, 41). This class of
proteins participates in tissue differentiation and cell proliferation.
Max binds to its cognate DNA with high affinity (~109
M1) as a homodimer in a scissors grip
fashion. The co-crystal structure of Max with its cognate sequence has
been solved by x-ray crystallography (38). To test our cross-linking
procedure, Max is a very good paradigm for specific DNA recognition.
RecA protein (molecular mass = 37.8 kDa) of Escherichia coli
participates in homologous recombination and DNA repair. In the
presence of ATP
S, RecA binds strongly to ssDNA (42). T7 RNAP (~99
kDa), which is normally a DNA-dependent RNA polymerase,
also binds ssDNAs (13, 43). Both RecA and T7 RNAP bind ssDNA without
sequence specificity and as such are very good candidates for testing
whether our cross-linking technique worked with nonspecific DNA-binding
proteins.
Fig. 2 shows cross-linking of Max to a 16-mer radiolabeled duplex DNA containing the cognate DNA-binding sequence (CACGTG).
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In the above experiment, 8-MOP (Fig. 1, I)
was added to DNA before the addition of Max. It was important to know
whether psoralen perturbed the binding of Max to DNA. We conducted a
series of experiments to show that Max binding to cognate DNA was
unaffected by the presence of 8-MOP. Fig. 4 shows a gel
mobility shift assay for Max binding to DNA in the presence or absence
of 8-MOP. A fixed concentration of DNA, either in the presence or
absence of 8-MOP was titrated with increasing concentrations of Max.
Binding of Max to the DNA was unaffected by the presence of 8-MOP. Fig. 3B shows binding isotherms expressed as fractional
saturation of DNA with increasing concentrations of Max. No difference
in the noncovalent binding affinity of Max to DNA was observed in the
presence of 8-MOP. At the lower Max:DNA ratios (<100 nM
Max), the binding isotherms were fitted to the equation of a straight line. The slope of the straight line was equivalent to the macroscopic binding constant of Max to the DNA (Fig. 3C). The
macroscopic binding constant (~109
M1) of Max to its cognate sequence was the
same with or without 8-MOP. This result showed that the presence of
8-MOP did not inhibit binding of Max. To verify that Max contacted the
E-box (CACGTG) in the presence of 8-MOP, we carried out DNase I
footprinting. Fig. 5, A and B,
shows a footprint of Max on 32P-labeled DNA. Within the
resolution of DNase I footprinting technique, the footprints of Max on
the E-box CACGTG are identical, with or without 8-MOP (Fig.
5B). These results demonstrate that the presence of 8-MOP
neither quantitatively (as measured by the binding constant) nor
qualitatively (as indicated by DNase I footprints) perturbed Max
recognition of its cognate DNA, indicating that cross-linking probably
occurred in a native Max-DNA complex.
Next, we examined if Max binding to cognate DNA was disrupted by the
addition of increasing amounts of 8-MOP. This information is essential
for maximizing cross-link yields in scale-up cross-link preparations
with the aim of determining the cross-linking sites on Max. One way to
maximize cross-link yields is to photochemically saturate the
cross-linkable sites in protein-DNA complexes. This can be achieved by
increasing the concentration of 8-MOP in the reaction. Clearly, this
cannot be achieved at the cost of binding specificity. Fig.
6 shows that Max binding to DNA was not inhibited even
at a 500- or 1000-fold excess of 8-MOP over DNA. In the lanes where
8-MOP was present (Fig. 6, lanes 1-6), Max-DNA complexes migrated as one band at the same position as when 8-MOP was absent (Fig. 6, lane 11), implying that identical complexes are
formed in the presence or absence of 8-MOP. At very high concentrations approaching saturating amounts of 8-MOP (36 µg/ml), Max binding to
DNA was inhibited by about 50-70% (Fig. 6, lanes 7 and
8).
Case 2, Nonspecific DNA Recognition: Cross-linking of Single-stranded DNA-binding Proteins to DNA
A 12-mer DNA
(5-GAAGCTACGAGC-3
) was furan-side monoadducted at the
unique dT. Fig. 7 shows a time course of cross-linking of E. coli RecA protein (Fig. 7A)
and T7 RNAP (Fig. 7B). The RecA cross-links migrated at
~42 kDa, while the T7 RNAP cross-links migrated at ~100 kDa,
consistent with the molecular masses of the proteins plus a 12-mer DNA.
The yield of cross-links increases with increasing UVA dose, indicating
that cross-linking is totally dependent on UV light (Fig.
8), in a manner similar to Max (Fig. 3A).
Under these irradiation conditions (Fig. 7) we have estimated that
20-60% of RecA or T7 RNAP was conjugated to DNA.
We have roughly estimated the quantum yield. represents mols of
cross-linked protein/the absorption cross-section (
a);
a = 2.303 (A/bn), where
A is the absorbance of the sample given by A = 1/2.303 ×
× (lnL0
lnLt);
for 8-MOP is 11.8 × 103
M
1 cm
1 at 300 nm; n
is Avogadro's number; b is the path length in cm. These
experiments were conducted using a monochromator attached to the
mercury/xenon arc source (see "Methods"). We measured the average
amount of light impinging the sample (L0) and
the average amount of light transmitted through the sample
(Lt) during irradiation. We assumed that only one
psoralen monoadduct reacted per RecA. The estimated quantum yield was
0.002-0.015 between 290 and 320 nm. The highest yield (0.015)
was obtained at 300 nm, consistent with our action spectra results (see
below). Quantum yields were not corrected for the binding
constant for the RecA/12-Maf interaction. This efficiency is somewhat
lower than those reported for photobinding of psoralens to DNA (44,
45).
For the isolation and purification of cross-linked peptides it is important to know the stability of the photoadducts (see Refs. 13 and 14 for method of adduct purification). We have tested the chemical stability of DNA-psoralen-protein conjugates against certain common laboratory reagents used during the course of conjugate purification. The adducts are stable to heat (~95 °C), 1-10% SDS, 8 M urea, 0.1% trifluoroacetic acid, 80% CH3CN, ethyl and methyl alcohols; phenol; 5% trichloroacetic acid; and modification by iodoacetamide. Some loss of DNA from the photoconjugate was observed after treatment with HCOOH (pH 1-2). Reversal of the DNA from protein was achieved by treatment with hot base (0.1 M KOH) at 95 °C or with 254-nm UV (photoreversal). This procedure is essential for the determination of the masses of psoralen-conjugated peptides (13, 14).
Wavelength Dependence of Photochemical Cross-linkingAction spectra provide two essential pieces of information regarding photochemical cross-linking: 1) action spectra can identify the most effective wavelength of UV at which optimal cross-link yield can be achieved; and 2) action spectra can reveal the differences in the mechanisms between protein-DNA cross-linking and DNA-DNA cross-linking.
Fig. 9 shows a spectral overlap of psoralen, DNA, DNA
Maf, and protein. DNA and protein effectively stop absorbing UV at
wavelengths above 310 nm. Psoralen furan-side monoadducts absorb UV
effectively at least up to 370 nm, and free psoralen absorption extends
to at least 410 nm. The absorption spectrum of psoralen shows two maxima: a larger maximum at ~250 nm ( = ~2.2 × 104 M
1 cm
1) and a
smaller maximum at 300 nm (
= ~1 × 104
M
1 cm
1). We examined the action
spectra for protein-DNA and DNA-DNA cross-linking via HMT from 220 through 380 nm. The measured absolute energy distributions of the
isolated UV bands are given under "Methods." The data represent the
average energies of UV passing through the empty cuvette slit recorded
during the course of the experiments. Here, it should be emphasized
that the monochromator, sample volumes, sample irradiation geometry,
band energy distributions, and total photon fluxes per experimental
sample at a given wavelength are the same. In addition, the same
protein amounts, DNAs, and psoralen were used. To minimize sample to
sample variation, we prepared stock irradiation mixtures from which the
same amount of sample was taken for irradiation at each wavelength, and
we kept the absorbance of the sample to a minimum. Each datum point in
Fig. 10 is an average of three separate sample
irradiations, and the error bars indicate minimal deviation.
These experimental procedures allow for reliable comparisons and a
straightforward interpretation of the action spectra.
Fig. 10, A-D, shows the relative action spectra for cross-linking DNA to DNA and DNA to protein (RecA) via HMT. The action spectra for cross-linking protein to DNA (Fig. 10, A and B) are significantly different from those for cross-linking DNA to DNA (Fig. 10, C and D). Cross-linking a 12-Maf to RecA protein has an action maximum at 300 nm (Fig. 10A, 1) coincident with one of the absorption maxima (~300 nm) for HMT (Fig. 9). An action maximum at 300 nm is also observed (Fig. 10B, 1) when free HMT is used to cross-link RecA protein to an unmodified 12-mer DNA. There is a smaller action peak at 365 nm (Fig. 10B, 2). For cross-linking a furan-side monoadduct to RecA, the 300-nm action peak 1 is about 3 times greater than the 365-nm action peak 2 (Fig. 10A), whereas when cross-linking is done with free HMT, the 300-nm action peak 1 is only ~1.7 times greater than peak 2 (365 nm). These differences are small but have been consistently reproduced. This result showed that protein-DNA cross-linking by HMT is most optimal at 300 nm compared with at 365 nm. When the 12-mer DNA is cross-linked to a complementary 8-mer DNA with free HMT or with a HMT furan-side monoadduct (DNA-DNA cross-linking), a different action spectrum is seen. Two features are apparently different: 1) the action maximum (Fig. 10, C and D, 1) is now red-shifted to 320 nm, compared with the action maximum 1 at 300 nm for DNA-protein cross-linking; and 2) the ratio of action peaks 1 at 320 nm to 2 at 365 nm (Fig. 10, C and D) is ~1.4.
At 365 nm, psoralen-DNA photocross-linking is favored over photoreversal. At 320 nm, both DNA monoadduction and DNA cross-linking occur (Fig. 10D). The action spectra show smaller peaks at 365 than at 320 nm, probably because of the lower psoralen extinction coefficient at 365 than at 300 or 320 nm (Fig. 9). The broad HMT absorption band (with an ~300-nm maximum; Fig. 9) is responsible for cross-linking. From a photochemical mechanistic view point, the significant feature is the apparent 20-nm blue shift for protein-DNA cross-linking compared with DNA-DNA cross-linking (see "Discussion").
Our present work was carried out with two specific aims: 1) to demonstrate the application of psoralen as a photocross-linking agent for studying specific and nonspecific DNA-protein recognition, and 2) to find the optimum UV wavelength for cross-linking and to move toward an understanding of the cross-linking mechanism. We have devised two complementary cross-linking approaches to the study of DNA-protein interactions: 1) for double-stranded DNA-binding sequence-specific proteins, using Max as a paradigm, and 2) for ssDNA-binding proteins, using RecA and T7 RNAP as examples.
Specific cross-linking of Max to its cognate sequence was achieved via
the addition of free 8-MOP. It may also be possible to cross-link Max
via furan-side monoadducts, just as with RecA and T7 RNAP. However,
since there are no favored 5-TpA sites within the E-box (CACGTG),
cross-linking via the use of monoadducts may yield less information.
Cross-linking via intercalated/stacked psoralen is advantageous because
there is no preselection of cross-linking sites. The whole gamut of
cross-linkable sites can be potentially examined by scale-up procedures
using free psoralen. In previously published works, we mapped
promoter-binding cleft and ssDNA binding finger in the
three-dimensional structure of T7 RNAP using the psoralen cross-linking
approach (13, 14). We hope to apply our mapping procedures to Max,
sterol receptor-binding protein, and other proteins. Cross-linking may
occur via the favored pyrimidines in and around the E-box. Direct
cross-linking with added free psoralen is a broad spectrum approach
that may yield structural information about global DNA-binding regions
in proteins. Irradiation of an intercalated psoralen furan-side
monoadduct will cross-link the dT in the complementary strand in
double-stranded DNA (13) more avidly than a bound protein, perhaps
because of geometric and quantum yield factors. Therefore, the
cross-linking with psoralen furan-side DNA adducts may be less useful
with sequence-specific double-stranded DNA binding proteins, mainly
because of competing DNA-DNA cross-linking and preselectivity of site.
However, this approach may be applicable with proteins that locally
unwind DNA at their binding sites, such as helicases, DNA-repair
proteins, and open complexes of DNA and RNA polymerases. Indeed, we
have earlier demonstrated that a T7 RNAP complex whose elongation was blocked by a site-specific psoralen monoadduct can be
cross-linked to the DNA template via the psoralen (13).
When psoralen is added to DNA followed by protein, there are at least five competing intermolecular photoreactions: psoralen-psoralen cycloadditions, DNA monoaddition, DNA furan-side monoadduct-DNA cross-linking, psoralen-protein reactions, and DNA-protein cross-linking. In addition, photodestruction and quenching by solvent molecules such as O2 also occur. The relative yields of these photoreactions are governed by a large number of factors that may include steric, kinetic, and thermodynamic factors; quantum yields of singlet and triplet excited states; and intersystem crossing. To compensate for the very large competition from the other reactions and to enhance our chances of trapping protein-DNA cross-links, we are using a large excess of psoralen. For Max cross-linking, a 1000-fold excess of 8-MOP over DNA was added. No detectable adverse effects on binding were seen by footprinting and gel shift assays. Cross-links are formed by the photoreaction of DNA-intercalated/stacked psoralen and bound protein. Photocoupling occurs through the absorption of two photons, most probably through two separate excitation events. The first absorbed photon probably leads to monoadducts with DNA. Absorption of a second photon only by furan-side DNA monoadducts leads to cross-linking with protein. Our estimated quantum yields are in the range of ~0.002-0.01, depending on the wavelength of UV. These quantum yields are smaller than those reported for the conversion of furan-side monoadducts to interstrand DNA cross-links (0.02-0.04) (46, 47). In our photoreactions, any Max that first reacted with free psoralen may not have cross-linked with DNA for the following reasons. 1) A specific stereochemical intercalation geometry is required for a (2 + 2) photocycloaddition to DNA, which is less likely to occur with a psoralen that was first photochemically attached to Max. However, it is possible that following "photochemical fixation" of Max to DNA by the first absorbed photon, other neighboring psoralens may photoreact with Max. 2) Photomodified psoralen that is first attached to protein via the pyrone side (3,4-double bond or C-2=O) will not absorb a long wave UVA photon to undergo a (2 + 2) reaction with DNA. The suggested overall pathway for cross-linking is as follows.
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Intercalation of psoralen changes the helical parameters of DNA (19,
48). 1) In both photochemical adducts, the psoralen is intercalated
between the base pair steps. 2) The repeat length is altered (overall
11-base pair repeat at the psoralen site as opposed to 10.5 base pairs
in standard B-DNA) consistent with the intercalation of psoralen. The
DNA returns to normal B form outside 3 base pairs of the intercalation
site of psoralen, and the helix parameters for both adducts conform to
normal B-DNA outside 3 base pairs of the intercalated psoralen. 3) The
normal Watson-Crick base pairing is preserved outside the intercalated site of the psoralen. 4) Hydrogen bonding of the base pairs above and
below the plane of the psoralen are not completely disrupted, although
in both adducts bases appear somewhat buckled. In the case of the
cross-link there may be increased solvent accessibility at the site of
the cross-link. 5) Overall, the DNA-helix has a very minor bend of
~8°. In furan-side monoadduct and cross-link, the helix is unwound
by ~27°-28°. 6) Due to the change in the hybridization of the
C-5-C-6 orbitals from sp2 to
sp3 following cycloaddition to dT, the
C-5-CH3 and C-6-H point away from the psoralen but still
point into the major groove. The 5 bases above and below the psoralen
stack onto the CH3 of the photoalkylated dT, coplanar with
the pyrimidine ring. 7) We estimated that psoralen monoadducts
cross-link to a protein that is within ~8 Å of the furan-side
adduct. One should note that the above description is for covalent
adducts of psoralen with DNA. Noncovalently bound psoralen probably has
a different configuration. It may be less rigidly bound within the DNA
and exchanges freely in solution. Because intercalators are generally
thought to obey the "nearest neighbor exclusion principle" (49),
noncovalently bound psoralens might be intercalated between every
second DNA base pair. Assuming a random mode of intercalation, at
equilibrium, there should be at least two sets of DNA molecules in the
population that have psoralen intercalated between alternating base
pairs (other combinations may also occur).
Cross-linking via furan-side monoadducts is more applicable to
ssDNA-binding proteins because they are in general nonspecific DNA-binding proteins, and therefore, the psoralen monoadduct can be
located at any convenient 5-TpA position within the ssDNA sequence
without altering binding. Moreover, in ssDNAs without self-complementarity, intercalation of psoralen (in the formal sense)
is unlikely to occur. Cross-linking is more likely to occur between DNA
and protein in the absence of the competing reaction with complementary
DNA strand.
What excited states might have participated in protein-DNA
cross-linking? Between 200 and 400 nm, psoralen shows two transitions (Fig. 11): an n
* transition
localized in the C-2 carbonyl and the
* localized in the
ring system (Refs. 15, 17, and 50 and references therein). The charge
transfer character of the 4
,5
-furyl double bond is lower than that of
the 3,4-pyronic double bond, and direct excitation of the furyl double
bond has not been observed (51). The lowest excited singlet and triplet states are the
* and are implicated in the (2 + 2)
cycloaddition with pyrimidines of nucleic acids (Fig. 11) (50, 52).
Based on molecular orbital calculations and luminescence spectra, the 3,4-double bond in the pyrone ring is the site for triplet (
,
*) reactivity (51, 53). Electronic, steric, and kinetic factors will
determine whether the 1
,
* or 3
,
*
participate in photoconjugation to DNA. It may be inferred that in
furan-side monoadducts (Fig. 1, III) only, the pyronic 3,4-double bond and/or the C-2=O are the photoreactive centers for
protein-DNA cross-linking. Because the furyl 4
,5
-double bond is
saturated by attachment to DNA (Fig. 1, III) it is probably taken out of action for protein cross-linking. Because the 290-320-nm absorption band (Fig. 9) is due to the coumarin in psoralen, the action
peak 1 (Fig. 10, A and B) perhaps represents
cross-linking via 3n,
* localized in the
carbonyl C-2=O (Fig. 11). Carbonyl n,
* states have some
,
* character in them, especially in polyaromatic molecules such
as psoralens because of mixing of states (54). The smaller peak 2 in
Fig. 10B may represent participation of
,
* transition (pyrone 3,4-double bond) in protein-DNA cross-linking at higher wavelengths (365 nm). The almost exclusive action peak (Fig.
10A, 1) for protein cross-linking with furan-side
monoadduct (suppression of peak 2; Fig. 10A) may be
attributed to the coumarinic pyrone C-2=O
(3n,
*). Thus, while 3
,
*,
localized in the olefinic 3,4-pyrone or 4
,5
-double bond of furan
participates in (2 + 2) cycloaddition to DNA and in DNA-DNA cross-linking (in agreement with previous reports), it is proposed that
the C-2=O (3n,
*) is predominantly involved in
the reaction with protein. In support for this suggestion, we observed
a 20-nm blue shift in the action maximum (Fig. 10, A and
B, 300-nm peak 1) for DNA-protein cross-linking
compared with DNA-DNA cross-linking (Fig. 10, C and D, 320-nm peak 1). This blue shift translated
into a higher potential energy of 8 kcal/mol or 0.212 eV for protein
cross-linking compared with DNA-DNA cross-linking. (In this context, it
is worth noting that the experimentally determined
E for
singlet-triplet splitting of n,
* states in carbonyl
compounds is generally 7-10 kcal/mol (54)). This implies that protein
cross-linking occurs at a higher potential energy surface
(3n,
*) as compared with DNA cross-linking
(3
,
*). This suggestion is in agreement with the
energy diagram (Fig. 11), which shows that
3n,
* is higher than 3
,
*.
(This does not mean that 3n,
* has 8 kcal/mol
higher energy than 3
,
*). The quantum yield for
singlet to triplet intersystem crossing is high, as evidenced by high
phosphorescence:fluorescence ratios (50-52). With most psoralens, the
triplet states are long lasting (up to 1 s) (34).
We thank Adrian R. Ferré-D'Amaré and Stephen K. Burley of the laboratory of molecular biophysics and the Howard Hughes Medical Institute for the supply of Max and cognate DNAs and suggestions. We thank Drs. Brian Chait, Steven Cohen, and Urooj Mirza for helpful discussions and Drs. Peter Model, David Mauzerall, William Agosta, Q. Lu, and Hal Lewis for advice and comments on the manuscript. We thank Prof. J. Lederberg for his interest in the project. Drs. Mauzerall and Agosta are thanked for the use of some instruments.