Photolyase-like repair of psoralen-crosslinked nucleic acids

Article abstract of DOI:10.1002/anie.201103611

Psoralen-derived photolesions are efficiently repaired by a photolyase-like mechanism. The removal of the interstrand crosslink by photoelectron injection confers control over biochemical processes by light (see picture). This paves the way to new, site-selective uncaging applications, as demonstrated with a primer extension assay.

Full text of DOI:10.1002/anie.201103611

DOI: 10.1002/anie.201103611
DNA Repair
Photolyase-like Repair of Psoralen-Crosslinked Nucleic Acids**
Thorsten Stafforst* and Donald Hilvert*
Psoralens are plant natural products that damage DNA and
RNA in a light-dependent manner.[1] After intercalating into
double-helical regions of nucleic acid, they react photochemi-
cally with thymidine or uridine residues to form cyclobutane
adducts via [2 + 2] cycloaddition reactions. At 5’-d(TA)
motifs, covalent interstrand crosslinks can result (Scheme 1).
Linking the two strands in this way prevents unpairing and
thymidine and the furan and pyrone psoralen moieties
(Scheme 1) resemble CPDs, their repair by a photolyase-
like mechanism has never been reported, nor has a psoralen-
specific photolyase been identified. Since light-triggered
release of psoralen crosslinks in DNA would be useful for a
variety of photocaging applications,[8] we have investigated
the feasibility of extending the PET strategy to the site-
selective repair of such adducts.
Electron-transfer reactions are strongly
distance-dependent, so the design of a suc-
cessful photolyase mimic requires a means of
binding an electron donor near the psoralen
crosslink.[9] For this purpose, peptide nucleic
acids (PNAs) are an attractive option. PNAs
are DNA mimics with a pseudopeptide back-
Scheme 1. Aminomethyltrisoralen (AMT) forms interstrand crosslinks with the 5’-TA
motif in duplex DNA through two consecutive [2+2] photocyclizations. The configuration
of the resulting crosslink is shown.[1b]
bone composed of neutral, achiral N-(2-ami-
noethyl)glycine units.[10] They hybridize with
high affinity and selectivity to complementary
sequences in single-stranded DNA and RNA
and can even invade double-stranded secon-
thus strongly impairs fundamental biological processes such
as replication[2] and transcription.[3] These properties have
made psoralens useful as probes of nucleic acid structure and
function and also as agents for the treatment of psoriasis and
other skin conditions.[1,4]
dary structures.[11] We therefore anticipated that a short PNA
bearing a PET chromophore would allow site-selective
delivery of the probe to a specific crosslink, thereby facilitat-
ing subsequent photorepair (Scheme 2). As electron donor
we chose a phenothiazine (Ptz) derivative rather than the
reduced flavin used by natural photolyases because it is more
efficiently excited and does not require in situ reduction.[12]
This chromophore was attached to an Fmoc-protected N-(2-
aminoethyl)glycine building block that is easily incorporated
into PNA oligomers by standard solid-phase peptide synthesis
(for details of the synthesis, see the Supporting Informa-
tion).[13]
We used a partially self-complementary DNA oligonu-
cleotide containing a single interstrand crosslink as the
substrate for the repair experiments (Scheme 2). It was
prepared by irradiating 5’-d(GCCTAGGCAGGCAAGC-
GAC) in the presence of AMT,[14] a commonly used psoralen
derivative, in neutral Tris-HCl buffer (50 mm, 10 mm MgCl2,
100 mm NaCl, pH 7.5) at (340 ꢀ 10) nm for 7 h at a constant
temperature of 48C. The crosslinked duplex was purified by
polyacrylamide gel electrophoresis (PAGE), and its identity
was confirmed by mass spectrometry (Supporting Informa-
tion, Figure S1). The sequence immediately downstream of
the crosslink is complementary to the PNA derivative
tgcctgcc-Ptz, so rapid hybridization ensues when the DNA
substrate (20 ngmLꢁ1 = 1.7 mm) is mixed with a 30% molar
excess of the PNA per target site in phosphate buffer (10 mm,
100 mm NaCl, pH 7.0). Following initial binding to the
exposed single-stranded nucleation site, strong PNA–DNA
interactions allow the PNA probe to invade the adjacent
duplex, even at physiological ionic strength, to place the
phenothiazine chromophore near the lesion (Scheme 2).
Repairing highly mutagenic psoralen crosslinks inside the
cell is a difficult and complex task that involves several repair
pathways.[5] In contrast, intrastrand cyclobutane pyrimidine
dimer (CPD) lesions that arise when DNA is exposed to UV
radiation are easily repaired either by nucleotide excision or
by a light-dependent, flavoenzyme-catalyzed process.[6] In the
latter case, photoinduced electron transfer (PET) from a
reduced and deprotonated flavin cofactor in the DNA
photolyase initiates [2 + 2] cycloreversion of the CPD, with
subsequent back electron transfer to the cofactor to regen-
erate the pyrimidine monomers. Photolyase action has been
successfully mimicked by simple model systems that position
a flavin chromophore proximal to a CPD or related DNA
lesion.[7] Although the cyclobutane rings formed between
[*] Dr. T. Stafforst, Prof. Dr. D. Hilvert
Laboratorium fꢀr Organische Chemie, ETH Zꢀrich
Wolfgang-Pauli-Strasse 10, Zꢀrich (Switzerland)
E-mail: thorsten.stafforst@uni-tuebingen.de
hilvert@org.chem.ethz.ch
Homepage: http://www.ifib.uni-tuebingen.de/forschung/
stafforst.html
http://www.protein.ethz.ch
[**] This work was generously supported by the Deutsche Akademie der
Naturforscher Leopoldina (Bundesministerium fꢀr Bildung und
Forschung BMBF-LPD 9901/8-158) and the ETH Zꢀrich.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201103611.
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Figure 1. PET-induced DNA repair. A) After hybridizing crosslinked
xlDNA (1.7 mm) with tgcctgcc-Ptz (4.5 mm) in 10 mm sodium phos-
phate, 100 mm NaCl, pH 7.0 buffer, samples were irradiated with
monochromatic light ((330ꢀ20) nm, 208C). Aliquots were removed
periodically, analyzed by denaturing 15% PAGE in 7m urea, and
visualized with SYBR Gold stain. xlDNA is cleaved to give a fully
uncaged oligonucleotide (ssDNA) and a psoralen monoadduct inter-
mediate with a half-life of 26 min, as determined by densitometry (see
Figure S3 in the Supporting Information). B) Whereas fully comple-
mentary wildtype (wt) tgcctgcc-Ptz repairs the crosslink, no detectable
photoreversal was observed in control experiments lacking PNA (—)
or with the mismatched sequence tgctcgcc-Ptz (mm). For estimation
of the repair yield, a dilution series (25–100 ng) of the starting material
(xlDNA) and the final product (ssDNA) were loaded on each gel.
Scheme 2. Strategy for photoactivating psoralen-crosslinked DNA. The
AMT crosslink (green) is introduced at a specific 5’-d(TA) motif in a
partly self-pairing DNA oligomer (top). A complementary PNA mole-
cule (blue) delivers an attached phenothiazine (Ptz) chromophore to
the lesion site (middle). Irradiation of the resulting complex at 330 nm
reverses the crosslink (bottom).
utility of psoralen caging/uncaging for controlling biochem-
ical processes with light. As outlined in Scheme 3, we
synthesized a 30 nucleotide (nt) long, partially self-comple-
mentary DNA oligonucleotide and crosslinked it with AMT
as described above. The crosslinked species was purified by
15% PAGE (7m urea) and then ligated at both 3’-ends to a
20 nt primer binding site using a splint ligation strategy (see
the Supporting Information). The product—a symmetric 2 ꢀ
50 nt construct, specifically crosslinked 36 nt downstream of
each 3’-terminus—was purified by 10% PAGE (7m urea) and
characterized by mass spectrometry (Supporting Information,
Figure S2). It served as the template for extension of a 5’-
FITC-labeled primer by the Klenow fragment (3’!5’ exoꢁ,
New England Biolabs). Since the polymerase cannot read
through the crosslink, an approximately 36 nt truncated
transcript is produced rather than the 50 nt run-off transcript
obtained from its uncaged counterpart (Figure 2). When the
crosslinked template was preincubated with the tgcctgcc-Ptz
PNA probe and irradiated at 330 nm, however, primer
extension yielded the full-length transcript as a result of
PET-induced cleavage of the psoralen linkage. The relative
amounts of truncated and full-length transcript that were
obtained depend on the irradiation time and are consistent
with the repair kinetics observed with the shorter template
After incubation for 15 min, we initiated photoactivation
by irradiating the sample at (330 ꢀ 20) nm with a 75 W xenon
arc lamp equipped with a 310 nm cut-off filter. At various
time points, aliquots (5 mL = 100 ng) were removed from the
mixture, and the progress of the reaction was monitored by
PAGE (Figure 1A). Densitometric analysis of the gels
showed that cleavage of the crosslink follows first-order
kinetics with a half-life of approximately 26 min (Figure S3 in
the Supporting Information).
The reaction initially afforded two products which were
PAGE-purified and shown by mass spectrometry to be a
psoralen monoadduct and the fully uncaged oligonucleotide
(Supporting Information, Figure S4). Upon extended irradi-
ation, the former is converted quantitatively to the latter,
highlighting the excellent uncaging properties of this system.
In control experiments, no repair was detected in the absence
of the Ptz-PNA or with mismatched PNA sequences such as
tgctcgcc-Ptz (Figure 1B). Efficient photoactivation evidently
requires site-specific hybridization of the Ptz-PNA with the
crosslinked DNA.[15]
A simple primer extension assay from a template
containing a single psoralen crosslink illustrates the potential
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Figure 2. Primer extension assay. The crosslinked template was incu-
bated with wt tgcctgcc-Ptz and irradiated with monochromatic light
((330ꢀ20) nm, 208C) for 15 to 420 min prior to addition of the
polymerase. As a control, primer extension was also carried out with
crosslinked (xl) and free single-stranded (ss) templates in the absence
of PNA and light. The resulting DNA products were separated by 15%
PAGE (7m urea). The amount of 50 nt long elongation product
increased with increasing irradiation times, whereas the short product
(ꢂ36 nt) gradually disappeared, indicating conversion of the xl into
the ss template. Densitometric analysis showed that the reaction
obeys first-order kinetics with a half-life of approximately 85 min
(Figure S5 in the Supporting Information). Conditions: see Refer-
ence [22].
like lesions are repaired or bypassed[17] restricts their utility
for photocaging purposes. In contrast, basic biochemical
processes can be completely blocked by site-selective incor-
poration of a single psoralen lesion into plasmids[18] and
chromosomes.[19] Employing an external PET chromophore
for the repair of psoralen lesions provides a simple and
selective means of restoring activity. Because PNA constructs
have been designed for high-affinity recognition of a wide
variety of single-stranded and double-stranded DNA and
RNA structures,[10,11] it should be possible to create PNA PET
probes that target virtually any psoralen lesion. Moreover,
simple Watson–Crick pairing can be used to steer repair to
specific lesions without affecting others, thus providing a level
of control not possible with standard caging approaches.
Extension of this strategy to other sequence-specific DNA
binders, such as triplex-forming oligonucleotides[20] and
polyamides,[21] and other chromophores, for example with
more red-shifted absorbances or two-photon-absorbance
capabilities, would further enhance its utility.
Scheme 3. Psoralen photocaging/uncaging applied to a primer exten-
sion assay. Top: A 50 nt template, specifically crosslinked 36 nt down-
stream of the 3’ terminus, was generated with a splint ligation strategy.
Bottom: After hybridization with a 5’-FITC-labeled primer (magenta,
FITC=fluorescein isothiocyanate), transcription from the crosslinked
template by the Klenow fragment results in premature transcription
arrest after approximately 36 nt. In contrast, the full-length 50 nt run-
off transcript is obtained after cleavage of the crosslink upon hybrid-
ization of the template with the Ptz-PNA (blue) and irradiation at
330 nm.
Received: May 26, 2011
Published online: August 29, 2011
Keywords: DNA repair · peptide nucleic acids · photoactivation ·
photolyases · psoralens
(Figure 2A). As expected, no detectable repair of the cross-
linked template was observed with the mismatched tgctcgcc-
Ptz-PNA (Supporting Information, Figure S6). As a conse-
quence, it should be possible to selectively repair lesions at
complementary sites in mixtures containing multiple cross-
links.
In conclusion, our results demonstrate the feasibility of
cleaving interstrand psoralen crosslinks by photoinduced
reductive electron transfer. This capability paves the way
for selective photocaging applications that cannot be realized
by currently available methods.[8] While mRNA, plasmids,
and other nucleic acids can be caged by stochastic modifica-
tion of nucleobases or backbone phosphates with multiple
photolabile protecting groups,[16] unwanted residual activity
and incomplete photoactivation limit the practicality of such
approaches. Similarly, the ease with which intrastrand CPD-
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