Selective detection and quantification of oxidized abasic lesions in DNA

Article abstract of DOI:10.1021/ja073014e

The C4′-oxidized abasic site (C4-AP) and 1,4-dioxobutane (DOB) are oxidized abasic lesions that are produced by a variety of DNA damaging agents. For instance, C4-AP accounts for～40% of the lesions produced by the antitumor agent bleomycin. These lesions exhibit interesting and unique biochemical and biological effects in DNA. We have developed a simple, selective method for detecting femtomole quantities of C4-AP and DOB in DNA. The method takes advantage of the selective reactivity of the 1,4-dicarbonyl groups in the lesions with a biotinylated probe. Pretreatment of the DNA sample with DNA polymerase β enables one to distinguish between C4-AP and DOB. The adducts are quantified using a fluorescent assay. This is the first method for quantifying these lesions that does not require degradation of the biopolymer. It will be useful for analyzing the levels of these oxidized abasic lesions in DNA. Copyright

Full text of DOI:10.1021/ja073014e

Published on Web 06/26/2007
Selective Detection and Quantification of Oxidized Abasic Lesions in DNA
Shanta Dhar, Tetsuya Kodama, and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218
Received May 8, 2007; E-mail: mgreenberg@jhu.edu
Oxidized abasic lesions are produced via a variety of DNA
damaging agents and exert distinctive effects on DNA repair and
replication.1,2 With the exception of 2-deoxyribonolactone (L) and
AP, abasic site (and DNA lesions in general) detection is typically
carried out using mass spectrometry, following digestion of the
Figure 1. Yields of adduct with C4-AP (1).
biopolymer, which in some instances is derivatized prior to
digestion.3-6 Selective tagging of DNA lesions offers the possibility
of rapid and sensitive lesion detection without the need for
completely degrading the biopolymer or expensive instrumentation.
We report a reagent, which when used in conjunction with DNA
polymerase â and an ELISA like fluorescence assay enables
selective quantification of the C4-AP and DOB oxidized abasic
lesions.7
C4-AP and DOB result from the initial oxidation of the C4′-
and C5′-positions of DNA, respectively. Very little is known about
the biological effects of DOB, although it is a potential source of
the DNA alkylating agent, butene dial.8,9 In contrast, C4-AP is
known to disrupt replication in E. coli and is a substrate for base
excision repair.10,11 C4-AP is a major component of DNA damage
produced by the antitumor agent bleomycin and is also generated
by the enediyne natural products, whereas DOB is formed in
reactions with neocarzinostatin and enediynes.12-14 Both lesions
are detected following γ-irradiation, presumably because of the
accessibility of the respective hydrogen atoms to the freely diffusible
hydroxyl radical.8,15 C4-AP can be detected in small DNA
fragments via gel electrophoresis following reaction with hydra-
zine.16 However, in order to quantify C4-AP or DOB produced
randomly in DNA, one typically stabilizes and/or derivatizes the
lesions prior to GC/MS analysis.8,12
Figure 2. Reaction of 5 (5 mM, pH 8.2, 55 °C, 4 h) with DNA containing
(A) C4-AP (1) or (B) DOB (2).
hydroxyl group was present (Figure 1). Consequently, 5, which
could be used in conjunction with a recently developed fluorescence
detection method for quantifying lesions was synthesized.7 We
anticipated that the two lesions could be distinguished by taking
advantage of the fact that DNA polymerase â (Pol â) excises DOB
but not C4-AP (Scheme 1, Figure 2).19
The reactivity of 5 with duplex DNA containing C4-AP (1) or
a ternary complex containing DOB (GOH indicates the 3′-hydroxyl
terminus of the oligonucleotide that flanks DOB, 2) was examined
using previously synthesized oligonucleotides containing the lesions
at defined sites.17,20 Optimum conditions for tagging C4-AP (82.3
( 0.1%) and DOB (74.8 ( 0.7%) were found to be 5 mM 5 at pH
8.2 and 55 °C for 4 h (Figure 2). Slightly higher adduct yields did
not justify using twice the amount (10 mM) of 5. No reaction was
detected between DNA containing AP or L lesions and 5 (data not
shown). The stereoisomeric mixture of tricyclic adducts were
The method described here takes advantage of a common
structural feature. The masked 1,4-dicarbonyl group in C4-AP and
DOB is unique to other lesions and offers the opportunity to develop
a reagent that would be useful for their selective detection. We
previously reported that DOB reacted with Tris to form a tricyclic
adduct in a reaction that is characteristic of 1,4-dicarbonyl
molecules.17,18 Reaction of two Tris analogues (3, 4) with C4-AP
(1) indicated that higher adduct yields resulted when a third
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10.1021/ja073014e CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. C4-AP formation as a function of peplomycin concentration.
Scheme 1. Fluorescence Detection of C4-AP and DOB Using 5
Figure 4. Quantification of C4-AP and DOB production by γ-radiolysis
of the 287 nt PCR fragment under (A) aerobic (B) anaerobic conditions:
with Pol â, blue; without Pol â, red.
Overall, this study indicates that 5 is a useful research tool for
selectively detecting two significant DNA lesions at femtomolar
levels. The method will enable one to utilize the C4-AP and DOB
abasic lesions as biomarkers.
Acknowledgment. We are grateful for generous support from
the National Institute of General Medical Sciences (Grant GM-
063028) and for a Japan Society for the Promotion of Science
Postdoctoral Fellowship for Research Abroad to T.K. We thank
Dr. Liang Xue for technical assistance and helpful suggestions,
Professor Hiroshi Sugiyama for peplomycin, and Dr. David M.
Wilson for DNA polymerase â as well as helpful comments on
the manuscript.
Supporting Information Available: Experimental procedures for
the synthesis and characterization of 5 and all other experimental
procedures; MALDI-TOF MS of DNA adducts and sample fluorescence
data. This material is available free of charge via the Internet at http://
pubs.acs.org.
characterized by MALDI-TOF MS.21 Treatment of 1 with Pol â
prior to reaction with 5 had no effect on adduct yield (Figure 2).
However, no adduct was detected in DOB (2) samples pretreated
with Pol â, indicating that combined use of the enzyme and 5 will
enable one to individually quantify these two lesions.
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Having established the fluorescent assay, 5 was used to measure
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