Hole migration is the major pathway involved in alkali-labile lesion formation in DNA by the direct effect of ionizing radiation

Article abstract of DOI:10.1021/ja0678931

Electron transfer via nucleobase hole migration has been extensively studied in recent years. Holes are produced in DNA via the direct effect of γ-radiolysis. However, the contribution of this pathway to DNA damage is difficult to discern because common p

Full text of DOI:10.1021/ja0678931

Published on Web 01/04/2007
Hole Migration is the Major Pathway Involved in Alkali-Labile Lesion
Formation in DNA by the Direct Effect of Ionizing Radiation
Hui Ding and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218
Received November 4, 2006; E-mail: mgreenberg@jhu.edu
Scheme 2. Synthesis of Hole Migration Probes
Ionizing radiation is the most commonly used nonsurgical method
for treating cancer, and DNA damage is the source of its cytotoxic
effects. DNA is damaged directly (the “direct effect”) and via
reactive species (e.g., hydroxyl radical, OH•) generated from water
(the “indirect effect”) by ionizing radiation.1 The direct and indirect
effects are believed to contribute approximately equally to DNA
damage and to produce a common spectrum of products from
overlapping sets of initially formed reactive intermediates. Nucleo-
base addition is the predominant pathway for OH•. In contrast, the
direct effect is sufficiently energetic to ionize the sugar, phosphate,
and nucleobase groups.1-3 The latter produces nucleobase holes,
whose migration in DNA has been the focus of extensive
investigation.4-7 Our goal was to determine how large a contribution
hole migration makes in the distribution of DNA damage resulting
from the direct effect of ionizing radiation.
Molecular probes that facilitate detecting excess electron transfer
or short circuit hole migration in DNA have been reported.8,9 We
designed a molecular probe (1, Scheme 1) to selectively detect hole
migration using gel electrophoresis. The trimethoxyphenyl group
was included in 1 to serve as a sink in which to localize a cation
radical (hole). Using established chemistry as a precedent, the corres-
ponding cation radical was expected to fragment to 5-formyl-2′-
deoxyuridine (fdU), an alkali-labile lesion.10-12 Monomeric 1 was
readily synthesized via Pd(0) coupling of the corresponding styrene
to 5-iodo-2′-deoxyuridine (Scheme 2). The vicinal diol was intro-
duced as a mixture of diastereomers via OsO4. The nucleoside’s
hydroxyl groups were silylated (3) prior to carrying out the osmyl-
ation reaction when preparing phosphoramidite 4, in order to
distinguish them from the vicinal hydroxyl groups. Oligonucleotides
containing 1 were synthesized via automated solid-phase synthesis
using commercially available reagents with minor modifications.13
Cyclic voltammetry measurements on 1 confirmed that the tri-
methoxyphenyl group provided a suitable driving force (E0(1) )
1.36 V versus E0(dG) ) 1.58 V) for hole localization, consistent with
that measured for other trimethoxyphenyl containing molecules.13-15
In addition, monomeric 1 met the requirements for utilization as a
probe for photoinduced electron transfer. The nucleoside was
unaffected by direct photolysis (350 nm), but HPLC and ESI-MS
analysis revealed that it was rapidly converted to fdU when
irradiated in the presence of the substituted anthraquinone (AQ)
previously used to inject holes in DNA.7,13
Having established the integrity of the oxidative fragmentation
process in 1, we sought to determine if this molecule functioned
as an efficient hole trap in DNA without disrupting duplex structure.
The TM of 5b (52.2 ( 0.7 °C) was only 1.8 °C lower than that of
5a (54.0 ( 1.2 °C), indicating that the substituent in 1 oriented
toward the major groove did not significantly perturb duplex
structure. The functionality of 1 as a hole trap in DNA was
examined qualitatively using 6 in which anthraquinone was used
as a hole injector.16 Evidence for the formation of fdU was gleaned
by the appearance of a gel shift product upon reaction of the
photolysate with a commercially available aldehyde reactive probe.13
Quantitative studies were carried out using 7a,b in which the
sequence was chosen to maximize the efficiency of hole injection
and migration (Figure 1a).16 Treatment of 7a with NaIO4 produced
an alkali-labile lesion at the original site of 1, consistent with the
formation of fdU via oxidation of the vicinal diol. However,
Scheme 1. Alkali-Labile Lesion Formation via Hole Trapping by 1
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10.1021/ja0678931 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Effect of sodium thiocyanate (NaSCN) on yield of alkali-labile
lesions in 7a upon exposure to 137Cs.
measure the small amounts of (background corrected) cleavage at
individual nucleotides other than 1.
Assuming that cleavage at 1 was attributable to direct ionization,
and that the indirect effect was responsible for alkali-labile lesions
at all other nucleotides, we estimated that the direct effect accounted
for at least two-thirds of the alkali-labile lesions produced in 7a
upon exposure to 137Cs. Support for mechanistically partitioning the
cleavage products in this manner was obtained by irradiating 7a in the
presence of the known OH• scavenger, sodium thiocyanate (Figure
2).18 Cleavage at 1 was at most modestly reduced due to competition
for the energy emitted by 137Cs. More importantly, cleavage at
nucleotides other than 1 was reduced to background levels. This
indicated that the indirect effect of γ-radiolysis was responsible
for the detected damage at these positions and not direct ionization
of the deoxyribose backbone or phosphate groups. From these data,
we conclude that alkali-labile lesions produced by the direct effect
of ionizing radiation on DNA are funneled to 1. Moreover, hole
formation/migration is the dominant component of the direct effect
of 137Cs that gives rise to alkali-labile lesions in solution.
Figure 1. Formation of alkali-labile lesions via hole trapping by 1. (A)
Photolysis (350 nm) of duplexes containing 1 with or without covalently
linked anthraquinone hole injector. (B) Use of 1 to detect hole migration
in 7a exposed to 137Cs. Lane 1, 150 Gy; Lane 2, 300 Gy; Lane 3, Fe•EDTA.
Lanes 1-3 were treated with piperidine.
Acknowledgment. We are grateful for support from the Nation-
al Institute of General Medical Sciences (GM-054996). We thank
Professor Gerald Meyer, Mr. James Gardner, Ms. Tamae Ito, and Dr.
Shanta Dhar for assistance with the cyclic voltammetry apparatus.
Table 1. Alkali-Labile Lesion Formation in 7a upon Exposure to
137Cs
trial
% 1 (#)a
% other (#)a,b
ratio 1:other
Supporting Information Available: Experimental procedures for
the synthesis/characterization of all molecules, and all other experiments.
HPLC, ESI-MS, phosphorimages from experiments described herein.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
2
3
9.1 ( 1.3 (9)
9.5 ( 0.8 (10)
7.9 ( 0.9 (10)
4.1 ( 1.3 (9)
4.1 ( 0.8 (10)
3.8 ( 1.2 (10)
2.2 ( 0.8
2.3 ( 0.5
2.1 ( 0.7
a # ) Number of replicates. b Other ) nucleotides other than 1.
References
piperidine labile sites were not detected following direct photolysis
(350 nm) of 7a. In contrast, photolysis of 7b produced an alkali-
labile lesion solely at the site of 1 in amounts proportional to the
irradiation time. Additional cleavage sites were not detected upon
treatment with base excision repair enzymes (Fpg, Endo III) that
excise damaged purines and pyrimidines.13,17 Any concern that 1
perturbed the duplex structure in such a way so as to favor its own
cleavage was dispelled by analyzing piperidine treatment of 7a
following exposure to OH• generated by Fe•EDTA (Figure 1b).
Overall, these data demonstrated that 1 was the sole repository of
damage following hole injection in 7b. Furthermore, the probe
provides a convenient readout for hole migration but does not bias
the reactivity of the primary DNA damaging reactive species (OH•)
produced by the indirect effect.
Hole formation by ionizing radiation (137Cs) was investigated in
7a. Irradiation of 7a generated an alkali-labile lesion at the site of
1 whose quantity varied linearly with dose.13 Although 1 was the
major piperidine labile site, alkali-labile lesions were formed in
small amounts at all other nucleotides in 7a.13 The ratio of alkali-
labile lesions produced at 1 relative to all other nucleotides in 7a
was determined in three separate experiments (Table 1). Multiple
replicates were carried out in each experiment in order to accurately
(1) Von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair;
Springer-Verlag: Berlin, 2006.
(2) Steenken, S.; Goldbergerova, L. J. Am. Chem Soc. 1998, 120, 3928-
3934.
(3) Purkayastha, S.; Bernhard, W. A. J. Phys. Chem. B 2004, 108, 18377-
18382.
(4) Delaney, S.; Barton, J. K. J. Org. Chem. 2003, 68, 6475-6483.
(5) Giese, B. Annu. ReV. Biochem. 2002, 71, 51-70.
(6) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res. 2001,
34, 159-170.
(7) Schuster, G. B. Acc. Chem. Res. 2000, 33, 253-260.
(8) Stafforst, T.; Diederichsen, U. Angew. Chem., Int. Ed. 2006, 45, 5376-
5380.
(9) Nakatani, K.; Dohno, C.; Saito, I. J. Am. Chem. Soc. 2001, 123, 9681-
9682.
(10) Gaillard, E. R.; Whitten, D. G. Acc. Chem. Res. 1996, 29, 292-297.
(11) Sugiyama, H.; Matsuda, S.; Kino, K.; Zhang, Q.-M.; Yonei, S.; Saito, I.
Tetrahedron Lett. 1996, 37, 9067-9070.
(12) Matsubara, M.; Masaoka, A.; Tanaka, T.; Miyano, T.; Kato, N.; Terato,
H.; Ohyama, Y.; Iwai, S.; Ide, H. Biochemistry 2003, 42, 4993-5002.
(13) See Supporting Information.
(14) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618.
(15) Jonsson, M.; Lind, J.; Reitberger, T.; Eriksen, T. E.; Mere´nyi, G. J. Phys.
Chem. 1993, 97, 11278-11282.
(16) Joy, A.; Schuster, G. B. Chem. Commun. 2005, 2778-2784.
(17) David, S. S.; Williams, S. D. Chem. ReV. 1998, 98, 1221-1261.
(18) Milligan, J. R.; Aguilera, J. A.; Nguyen, T.-T. D. Radiat. Res. 1999, 151,
334-342.
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