DNA Damage and Mutagenesis
Purines as targets for DNA photodamage
R. J. H. Davies
School of Biology and Biochemistry, Medical Biology Centre, Queen's University, Belfast BT9 7BL, N. Ireland, U.K.
Introduction
UV radiation is arguably the most widely studied
Many different chemical compounds are capable
of acting as photosensitizers towards DNA [5].
They encompass some endogenous cellular
molecules, such as tlavins and porphyrin derivatives, as well as a wide variety of xenobiotics
including certain drugs and environmental pollutants. Usually, light absorption generates the
excited triplet state of the photosensitizer and
this constitutes the reactive species. Except in a
minority of cases, where the triplet excitation
energy is transferred directly to DNA or a simple
photoaddition reaction takes place, photosensitizers normally damage DNA through photo-oxidation. Because guanine is the most readily
oxidized of the DNA bases it tends to be preferentially modified or destroyed [2,6]. Adenine is,
by contrast, quite stable towards most photosensitizers. Photo-oxidation occurs via two main
mechanisms. In type-I processes, there is an
electron or hydrogen atom transfer between the
sensitizer and purine base. With guanine, the
transfer of an electron to the excited photosensitizer will generate the guanine radical cation as
the primary reactive intermediate. In type-I1 processes, the sensitizer transfers energy to molecular oxygen to yield singlet oxygen which
subsequently oxidizes the guanine nucleus. Both
pathways lead to the production of 7,8-dihydro8-oxo-2'-deoxyguanosine
in
native
DNA,
together with other oxidation products, which
collectively act as alkali-labile sites for DNA
chain cleavage [6-91. Treatment of photo-oxidized DNA with hot piperidine characteristically
induces preponderant strand scission at guanine
residues; this property has been exploited in a
chemical sequencing protocol for locating guanine bases in DNA [lo].
Mutagenicity studies have confirmed that
guanine is the major target for DNA modification caused by photo-oxidation. T h e formation of
7,8-dihydro-8-0~0-2'-deoxyguanosine leads predominantly to G-T transversions in bacterial
and mammalian systems [ 111.
Abbreviations used: AA* and A=A, dimeric adenine
photoproducts; 8-AIA, 8-(5-aminoimidazol-4-yl)adenine; DGPY, 4,6-diarnino-5-guanidinopyrimidine;
6-MIP, 6-methylimidazo[4,5-b]pyridin-5-one; TA*,
thymine-adenine photoadduct; UVA, radiation of
wavelength 320-400 nm; UVB, radiation of wavelength
280-320 nm; UVC,radiation of wavelength <280 nm.
T h e absorption of UVB and UVC photons by
cellular DNA molecules is well known to
have mutagenic and cytotoxic consequences
[2,3,12,13]. Much research has been devoted to
isolating and identifymg the underlying photo-
of all mutagenic agents, not least because of the
established link between solar UVB rays and
human skin cancer [l]. Its mutagenic and cytotoxic properties derive ultimately from photochemical modifications to the structure of DNA
[2-41 which, if they are not repaired, interfere
with the correct expression and transmission of
genetic information. This article briefly reviews
how UV light can damage the purine nucleobases
of DNA, with particular reference to the photoaddition reactions of adenine characterized by my
laboratory.
When considering the role of the purine
bases, adenine and guanine, as targets for UV
radiation damage in DNA, it is important to distinguish between photoreactions arising from the
direct absorption of photons by DNA and those
caused by photosensitization. T h e former are
largely intramolecular in nature whereas photosensitization involves the participation of other
molecules in electronically excited states. In
practice, the type of photoreaction that occurs
will depend critically on the wavelength of the
incident radiation. T h e patterns of photoreactivity associated with the conventional subdivisions
of the UV spectrum are essentially as follows.
Damage caused by UVC wavelengths (<280 nm)
is produced through direct absorption of the
radiation by DNA. In contrast, UVA wavelengths
(320-400 nm) beyond the absorption envelope of
DNA can only damage it through the agency of
photosensitizer molecules which absorb the light
instead. Photons with intermediate wavelengths
in the WB range (280-320 nm) can modify
DNA through either or both of these mechanisms depending on the experimental conditions.
Photosensitized damage
Direct excitation
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lesions. It is now clear that, in the absence of
photoionization [ 141 (induced by wavelengths in
the region of 200 nm), the main targets for UV
radiation damage are doublets of adjacent pyrimidine bases on the same strand of DNA. They
undergo photoaddition reactions to give either
cyclobutane dimers or pyrimidine (6-4) pyrimidone photoadducts [2,3]. In comparison with
thymine and cytosine, their purine counterparts
in DNA are very resistant to photochemical alteration under conditions of direct excitation. Guanine, in particular, is essentially inert and no
intramolecular photoproducts derived from it
have yet been isolated from UV-radiated DNA.
We have found, however, that adenine is capable
of forming photoadducts with adjacent thymine
or adenine bases on the same strand of DNA,
albeit in very low yield. The nature of these
photolesions is now discussed.
formation of the parent photoproduct TA* [19].
For native DNA this is in the region of 14 pmoV
einstein compared with a value of about 2500
pmoVeinstein for pyrimidine photodimerization
[14,20]. TA* is therefore a rare photoproduct in
DNA but it does appear to be highly mutagenic.
Zhao and Taylor [21] have successfully introduced a site-specific TA* photoadduct into the
(-)-strand of the replicative-form DNA of an
M13mpl8-derived phage. After replication in a
repair-deficient Escherichia coli host under SOS
conditions, 82% of the (-)-strand progeny
analysed were mutated. Replacement of the
original TA doublet by TT was the most frequent change. Thus, unless they are efficiently
repaired, TA* photolesions may play a minor role
in UV-induced mutagenesis.
Dimeric adenine photoadducts
Thymine-adenine photoadduct
The thymine-adenine photoadduct, designated
TA*, is produced when the dinucleoside monophosphate d(TpA) is irradiated at 254 nm in
aqueous solution [15]. It was originally inferred
from spectroscopic analysis to incorporate a
cyclobutane ring linking the C-5 and C-6 atoms
of thymine to C-6 and C-5 of adenine respectively [16,171. More recently, evidence derived
from I3C NMR strongly suggests [18] that it
exists as a less strained valence isomer of this
structure (see Figure 1). Sensitive detection of
TA* in UV-irradiated DNA is possible because
acid hydrolysis converts it specifically into the
intensely fluorescent heterocyclic base 6-methylimidazo[4,5-b]pyridin-5-one (6-MIP), depicted in
Figure 1. By measuring the amount of 6-MIP
present in acid hydrolysates of UV-irradiated
DNA, we have determined the quantum yield for
fisrure
'
Structures of the thymine-adenine photoadduct, TA*.
and its acid hydrolysis product 6-MIP
H
O\
Volume 25
T A'
6-MIP
Through studies on the model dinucleoside
monophosphate d(ApA), we have demonstrated
that photodimerization of adjacent adenine bases
in DNA occurs by cycloaddition of the N-7-C-8
double bond of the S'-adenine across the C-6
and C-5 positions of the 3'-adenine [22,23]. The
primary azetidine photoproduct thus formed is
chemically unstable and decomposes by competing reaction pathways to yield two distinct photoadduct species designated AA* and A=A (see
Figure 2). The presence of these photoadducts
in UV-irradiated DNA can be detected by virtue
of their respective conversion into the diagnostic
compounds
8-(5-aminoimidazol-4-yl)adenine
(8-AIA) and 4,6-diamino-5-guanidinopyrimidine
(DGPY) on acid hydrolysis (Figure 2).
We have used reverse-phase HPLC to isolate and quantify the amounts of 8-AIA and
DGPY found in acid hydrolysates of E. coli DNA
after irradiation at 254 nm [24,25]. T o attain sufficient sensitivity, the DNA was radiolabelled
with tritiated deoxyadenosine by nick translation
[25]. Individual quantum yields for the formation
of AA* and A=A can be calculated on the basis
of the amounts of recovered 8-AIA and DGPY
and summed to provide an overall quantum yield
for adenine photodimerization. The value for
denatured DNA thus obtained was in the range
60-120 pmolleinstein compared with 10-40
pmolleinstein for native DNA [25]. This implies
that the photodimerization reaction is markedly
quenched by base-pairing, as also observed [ 191
in the case of thymine-adenine photoaddition to
give TA*. Currently, no information is available
DNA Damage and Mutagenesis
on the mutagenicity of the dimeric adenine photoadducts, or their recognition and removal from
DNA by repair enzymes. However, as their combined yield in native DNA is estimated to be at
least 100 times lower than that of the major pyrimidine photoproducts they are probably of very
limited biological significance.
Conclusions
The purine bases in DNA show contrasting photoreactivity under conditions of direct excitation
and photosensitization. Guanine is the most susceptible of all the nucleobases towards photooxidation but adenine is relatively unaffected.
When double-stranded DNA is excited by the
Figure 2
Structures of the primary adenine photodimer, the derived stable photoadducts AA* and A=A,
acid hydrolysis products 8-AIA and DGPY
and their respective
Adonino Photodimor
/
\
0
\
1H+
lH+
DGPY
8-AM
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Biochemical Society Transactions
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absorption of photons at UVB and UVC wavelengths, guanine is very resistant to modification
but adenine can undergo photoaddition reactions
with neighbouring thymine or adenine bases.
T h e s e reactions occur with very low quantum
yields in the region of 20 PmoVeinstein. Consequently, the resulting TA*, AA* and A=A photoadducts should constitute no more than about
2% of the total photolesions produced in native
DNA by direct excitation; they are therefore
likely to be of marginal biological importance.
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Received 5 August 1996
Oxidative DNA damage in human cells: the influence of antioxidants and DNA
repair
A. R. Collins, S. J. Duthie, L. Fillion, C. M. Gedik, N. Vaughan and S. G. Wood
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 I 9SB, Scotland, U.K.
Introduction
O n e of the most important insults to which cellular DNA is subjected is oxidative damage,
resulting from attack by reactive oxygen species,
i.e. free radicals, of exogenous or endogenous
origin. Oxidative damage is implicated in the
Abbreviations used: FPG, formamidopyrimidine
glycosylase; GC-MS, gas chromatography with
mass spectrophotometric detection; 8-OH-dG,
7,8-dihydro-8-oxodeoxyguanosine; SCGE, single-cell
gel electrophoresis.
Volume 25
earliest stages of carcinogenesis [ 11. Epidemiological evidence linking the high incidence of
certain cancers with a low intake of fruit and
vegetables [Z-61 can be explained, at least in
part, by the presence in these foods of various
antioxidant micronutrients (vitamin C, carotenoids, vitamin E, flavonoids and other polyphenolics), which are believed to decrease the
amount of free radicals - particularly hydroxyl
radicals - reaching the DNA. Attempts to test
this hypothesis of antioxidant protection by
means of intervention trials have had mixed