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Sharp Kink of DNA at Psoralen-cross-link Site Deduced
from Crystal Structure of Psoralen-Thymine Monoadduct
S.-H. Kim, S. Peckler, B. Graves, et al.
Cold Spring Harb Symp Quant Biol 1983 47: 361-365
Access the most recent version at doi:10.1101/SQB.1983.047.01.042
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Copyright © 1983 Cold Spring Harbor Laboratory Press
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Sharp Kink of DNA at Psoralen-cross-linkSite Deduced from
Crystal Structureof Psoralen-Thymine Monoadduct
S.-H. KIM, S. PECKLER, B. GRAVES, D. KANNE, H. RAPOPORT, AND J . E . HEARST
Department of Chemistry and Lawrence Berkeley Laboratory, University of Californio, Berkeley, California 94720
Psoralens are a class of tricyclic aromatic heterocycles that have been used as chemical probes to study
DNA and RNA structures (for review, see Hearst
1981). On exposure to UV light, psoralens form chemical cross-links to the DNA bases in three steps (Isaacs et
al. 1977; Johnston et al. 1977). The first step is intercalation of psoralen between two adjacent base pairs. The
second step is formation of a monoadduct, i.e., one
psoralen photoreacts to one strand of DNA. The third
step is the cross-linking of the same psoralen to the other
strand of DNA.
Light-induced cross-linking of double-stranded nucleic acids by psoralens has been exploited to locate, in
vivo or in vitro, those double-helical regions of DNA or
RNA that can accommodate any structural changes
caused by the psoralen cross-links. To determine threedimensional structural parameters of the cross-link, we
have solved the crystal structure of the psoralen-thymine
monoadduct formed in photoreaction between calf
thymus DNA and 8-methoxypsoralen (8MOP).
Figure 1 shows the chemical structures and numbering system used for 8MOP and thymine. The photoaddi-
tion occurs at the double bonds between C 12 and C 13 of
the furan ring (site I) and between C3 and C4 of the
pyrone ring (site II). The preferred target is the double
bond at TC5 and TC6 of thymine, forming cyclobutane
rings by reacting at either site I or site II of the psoralen.
There are eight possible configurations for psoralenthymine monoadducts (Fig. 2) and 64 for diadducts.
Spectroscopic and other studies have narrowed this
down to a few possible configurations, and recent nuclear magnetic resonance (NMR) studies on psoralen monoadducts (Straub et al. 1981; Kanne et al. 1982b) and
crystallographic studies described here prove unambiguously that the biologically relevant structures have
the "cis-syn" configuration. Such drastic reduction in
the number of configurations comes from the fact that
the double-helical DNA conformation imposes very
stringent restrictions on modes of psoralen interaction.
Thus, presumably, only a particular configuration can
be formed.
We describe here the structural details of a psoralenthymine monoadduct obtained in a biological environment and the consequences of the photo-cross-link between 8MOP and double-helical DNA.
E X P E R I M E N T A L PROCEDURES
H15a . / /
f
011
~
The monoadduct of 8-methoxypsoralen-thymine
(8MOP-T) was synthesized and purified as described
below (Kanne et al. 1982b). 8MOP was added to DNA
and then irradiated with UV light at 320-380 nm. The
psoralen-DNA solution was extracted with chloroform
to remove unreacted psoralen and its photodegradation
products. This DNA-psoralen photoproduct was precip-
H15C
014
/
~ no
" H4
"[H1
cis-syn
9o2%
TH3
I
cis-anti
trans-syn
trans-anti
T.0
TN3
TO4
I~TC5
Figure 2. Nomenclatureand schematic representation of the eight
possible configurational isomers for 8MOP-thyminemonoadduct.
The large slabs represent 8MOP with the five-memberedfuran ring
at the left (indicated by solid rectangles). Handles projecting out
from these slabs represent the methoxygroup at C8 of the psoralen
ring. The smaller slabs are for thymine bases with the handles
representing TN1-TH1. The isomers on the top row are mirror images of the isomers on the bottom row. Two configurationsfound
in the crystal structures are cis-syn.
TH7c
TH7a
F~ure 1. Numberingsystem for 8MOP and thymine used in this
paper.
361
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362
KIM ET AL.
Figure 3. Stereodrawing of a single 8MOP-T molecule.
itated, acid-hydrolyzed, and purified by reversed-phase
and high-performance liquid chromatography.
8MOP-T was dissolved in a 60% aqueous methanol
solution in a partially airtight container and evaporated
extremely slowly for a period of 4 weeks. Well-formed
crystals were obtained that had a triclinic space group
with cell parameters a = 11.30 .~,, b = 14.05 A , c =
15.53 ~,, a = 7 5 . 6 2 0 , ~ = 8 7 . 0 9 0 , 7 = 8 4 . 2 6 ~ This
limits the possible choices of the space group to P1 and
P1. Eventual structure solution and refinement confirms
the latter space group to be the correct one. The unit cell
contains six molecules of 8MOP-T and two molecules
of methanol. The X-ray diffraction data were collected
on an automatic diffractometer using graphite-monochromated MoKc~ radiation. A total of 6079 reflections
have been collected using the 0-20 scanning mode with
variable scan rates. The crystal structure was solved by
the direct method (Main et al. 1980), and subsequent
refinement revealed the entire structure, including all
hydrogen atoms and methanol molecules. The entire
structure was refined anisotropically by the full-matrix
least-squares method. The final discrepancy factor (R
factor) is 5.2 %. A stereodrawing of one of three unique"
molecules is shown in Figure 3. Crystallographic details
are published elsewhere (Peckler et al. 1982).
RESULTS AND CONCLUSIONS
Psoralen Photoreacts to Both A-T and T-A
Sequences in DNA
In the unit cell there are three 8MOP-T molecules of
one-handedness, and the other three have other-handedness. This suggests that 8MOP can form photomonoadducts to an A-T sequence as well as T-A sequences in
double-helical DNA (see Fig. 4). Although two isomers
are present in a 1:1 ratio in crystals, isolation results
suggest that the ratio may be 3:2 in the hydrolysate
(Kanne et al. 1982b).
Angle between Planes of Psoralen and Base
Is Variable
The difference among the three unique molecules in
the unit cell is best manifested by the interplanar angle
between the psoralen and thymine moieties, as shown in
Figure 5. The interplanar angle ranges between 44.11 ~
c(53.5 ~
/
,, B(50.6")
5'
3'
5'
A ...........
3'
"
A ............ T
J
3'
5'
3'
5'
Figure 4. (Top) Schematic drawing to show the intercalation and
8MOP-T monoadductformation at 5 ' . . . T - A . . . 3 ' and 5 ' . . . A T... 3' sequences. (Bouom) Schematic drawing of two diadduet
enantiomers of c/s-syn configuration. The representations are the
same as those in Fig. 2.
THYMINE
Figure 5. Interplanarangles between thymine and 8MOP plane in
three unique 8MOP-T molecules.
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PSORALEN-THYMINE MONOADDUCT
/
//
/
JJl
363
and accommodated in double-helical DNA. This unique
diadduct had the cis-syn configuration at the pyrone
ring, as shown in Figure 4. This is consistent with the
observation by Kanne et al. (1982a).
Psoralen Cross-linking Introduces a Sharp Kink
in DNA Structure
(A-T)-8M0P-(T"Ai~'" ,~\
\
\
\
\
Figure 6. Schematic drawing of double-helical B-DNA (cylinders) linked by psoralen photo-cross-links. The T'A base pairs on
both sides of psoralen moietyare shown as rectangular plates. This
is based on a detailed molecular model built using skeletal atomic
components.
and 53.45 ~ This suggests that the interplanar angle is
not fixed but has a limited range of flexibility, with its
values depending on the environment. This, in turn, implies that psoralen-cross-linked DNA can still have
limited flexibility at the site of the cross-link.
Cis-Syn Configuration on
Both Sides of
Psoralen Plane
The configuration found in this crystal structure (cissyn) allows one to build molecular models for an
8MOP-T2 diadduct, where another thymine forms a
photoadduct at the pyrone ring. Our model building
shows that for each 8MOP-T monoadduct enantiomer,
there is only one 8MOP-T2 diadduct that can be built
As pointed out earlier, the angle between the thymine
and psoralen moieties in the structure ranges from 44 ~
to 53 ~ when the cyclobutane ring is formed at the furan
ring. If one assumes similar geometry at the pyrone
ring, then one can easily see that the extent of distortion
to the DNA that would be induced by psoralen crosslinking is substantial. Molecular model building based
on this structure suggests that the DNA may kink as
much as 70 ~ Psoralen cross-linking will not only unwind DNA, as was previously shown by the superhelical density studies (Weisehahn and Hearst 1978), but
also introduce a very sharp kink at the site where
psoralen is introduced (Figs. 6, 7, and 8). This kind of
distortion is important in that any portion of duplex
DNA that cannot accommodate psoralen intercalation or
a sharp kink will not form psoralen cross-links. This
line of reasoning leads one to conclude that the psoralenDNA photo-cross-linking may occur only in those
regions of duplex DNA that are flexible and are not constrained by proteins or a three-dimensional structure
(Hanson et al. 1976).
The sharp kink of the model suggests that, after the
monoadduct formation, there must be a drastic conformational change to form a kink before the second
photoreaction can occur to complete the cross-linking.
The necessity for the kink provides a possible explanation for the observed relaxation time of 1 #sec between
the monoadduct and formation of the diadduct (Johnston
et al. 1981). This is shown schematically in Figure 7.
Recently, Land et al. (1982) published a short communication reporting the structure of a photoadduct obtained by UV irradiation of a mixture of 8MOP and
thymine in an ice-methanol matrix in the presence of
benzophenone. Although this adduct was made under
nonbiological conditions without the structural constraints of the double-helical DNA and was presumably
one of many products formed in the reaction, the structure they obtained is the same as that reported here.
psoralen
hv
Figure 7. Presumed steps in psoralen-DNAcross-linking.
hv
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Figure 8. Molecular model of 8MOP-DNA cross-link.
364
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PSORALEN-THYMINE MONOADDUCT
ACKNOWLEDGMENTS
We thank Drs. Steve Holbrook and Fred Hollander
for their help in computations and data collection,
respectively. The work reported here has been supported by grants from the National Institutes of Health
(CA-27454 and GM-25151), the National Science
Foundation (PCM80-19468), and the Department of
Energy.
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