Free Radical Biology & Medicine, Vol. 33, No. 1, pp.

114, 2002
Copyright 2002 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/02/$see front matter

PII S0891-5849(02)00827-4

Serial Review: Oxidative DNA Damage and Repair

Guest Editor: Miral Dizdaroglu
BIOLOGICAL CONSEQUENCES OF FREE RADICAL-DAMAGED DNA BASES
SUSAN S. WALLACE
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, The University of Vermont,
Burlington, VT, USA
(Received 11 December 2001; Revised 12 February 2002; Accepted 1 March 2002)

AbstractThe principal oxidized cytosine bases, uracil glycol, 5-hydroxycytosine, and 5-hydroxyuracil, are readily
bypassed, miscode, and are thus important premutagenic lesions. Similarly the principal oxidation product of guanine,
8-oxoguanine, miscodes with A and is a premutagenic lesion. Most of the thymine and adenine products that retain their
ring structure primarily pair with their cognate bases and are not potent premutagenic lesions. Although thymine glycol
pairs with its cognate base and is not mutagenic it significantly distorts the DNA molecule and is a lethal lesion. Ring
fragmentation, ring contraction, and ring open products of both pyrimidines and purines block DNA polymerases and
are potentially lethal lesions. Although these breakdown products have the potential to mispair during translesion
synthesis, the mutational spectra of prokaryotic mutants defective in the pyrimidine-specific and/or purine-specific DNA
glycosylases do not reflect that expected of the breakdown products. Taken together, the data suggest that the principal
biological consequences of endogenously produced and unrepaired free radical-damaged DNA bases are
mutations. 2002 Elsevier Science Inc.
KeywordsFree radical DNA damage, Oxidized pyrimidines, Oxidized purines, Base excision repair, Oxidized bases
and DNA polymerases, Oxidized bases and mutations

INTRODUCTION

lethal lesion. If, on the other hand, the lesion is not

structurally distorting, pairs with a noncognate base and
is readily bypassed by DNA polymerases, it is taken to
be premutagenic. Some lesions are neither mutagenic nor
lethal while others are lethal and mutagenic by virtue of
being able to be bypassed during translesion synthesis by
a specialized polymerase. To verify biochemical predictions, individual lesions have been introduced into unrepairable single-stranded viral DNA molecules and assessed for their lethal and mutagenic potential [3 6].
This approach is advantageous because lesion structure
and in vitro interactions with DNA polymerases can be
directly related to biological consequences; the disadvantage is that only a single sequence context is usually
investigated. Finally, sensitivity to DNA-damaging
agents and mutational spectra determined in DNA repair
mutants defective in the processing of individual lesions
give further support to potential lethality and mutagenicity of those lesions. The disadvantage here is that multiple lesions are present in the DNA at the same time.
Taken together, however, these data have provided the
underpinning for our understanding of the biological

The biological consequences of individual damaged

bases has been deduced by a combination of experimental approaches. Structural studies of individual model
lesions have provided information as to the potential
coding properties of that damage and whether it is structurally distorting to the DNA molecule. In vitro, biochemical studies with lesions randomly (older studies) or
site-specifically into template DNA molecules have been
employed to assess their interactions with various DNA
polymerases (for reviews see [1,2]). If the lesion is
structurally distorting and blocks the progression of the
DNA polymerase molecule, it is taken as a potentially
This article is part of a series of reviews on Oxidative DNA
Damage and Repair. The full list of papers may be found on the
homepage of the journal.
Susan S. Wallace, Ph.D., is Professor and Chair of the Department of
Microbiology and Molecular Genetics at the University of Vermont.
Address correspondence to: Dr. Susan S. Wallace, Department of
Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, The University of Vermont, 95 Carrigan Drive, Stafford
Hall, Burlington, VT 05405-0068, USA; Tel: (802) 656-2164; Fax:
(802) 656-8749; E-Mail: swallace@zoo.uvm.edu.
1

S. S. WALLACE

consequences of free radical-induced DNA base damages.

FREE RADICAL-DAMAGED THYMINE BASES

Historically the most well-studied oxidized pyrimidine base is 5,6-dihydro-5,6-dihydroxythymine (thymine

glycol). Thymine glycol is the most common thymine
lesion found after treatment by oxidizing agents such as
hydrogen peroxide, ionizing radiation, potassium permanganate, and osmium tetroxide [79]. Thymine glycol
exerts significant distortion on the duplex DNA molecule. Proton NMR spectroscopy on a duplex oligonucleotide containing thymine glycol shows that the base
portion of thymine glycol and the adenine opposite appear to be extrahelical [10]. Molecular dynamics simulations of 5-hydroxy-5,6-dihydrothymine, 6-hydroxy-5,6dihydrothymine, 5,6-dihydrothymine (dihydrothymine),
and thymine glycol support the idea that the hydroxy group
at the 5 position causes a pseudo-axial oriented methyl
group that results in distortion of the base pair 5 to both the
thymine glycol and the 5-hydroxy-5,6-dihydrothymine [11,
12]. In fact, primer extension studies with several DNA
polymerases using a template containing thymine glycol
show that extension occurs until the lesion site with adenine
inserted opposite the thymine glycol [1316]. In the presence of proofreading, extension can proceed no further,
supporting the idea that it is the base pair 5 to the thymine
glycol that is disturbed and suggesting that thymine glycol
is a lethal lesion. Further support for the conclusion from
the molecular dynamics calculations comes from studies
showing that 5,6-dihydro-5-hydroxythymidine is also a
strong block to Escherichia coli DNA polymerase I Klenow
fragment (exo-) [17]. As predicted from the biochemical
studies, thymine glycol is lethal having an inactivation
efficiency of one in biologically active single-stranded
DNA [3,5]; thymine glycol is also lethal in duplex DNA
[4,18]. There are, however, several sequence contexts
where thymine glycol is bypassed by certain DNA polymerases in vitro [16,19] and in vivo [20].
Because thymine glycol pairs with adenine it is a poor
premutagenic lesion. In SOS induced E. coli where translesion synthesis is upregulated, no mutations are found at
thymine glycol sites [5]. However, when thymine glycol
is inserted into a bypass sequence, a low level of misincorporation of G opposite thymine glycol occurs, giving
rise to mutation [20].
Another ring saturation product of thymine, dihydrothymine, has been well studied. Dihydrothymine does
not inhibit DNA polymerases [21] and it correctly pairs
with cognate adenine; accordingly it is neither lethal nor
mutagenic [6].
The 5-methyl oxidation products, 5-hydroxymethyluracil (5-HMU) [9,22,23] and 5-formyluracil (5-fU) [24]

are also formed by free radicals in DNA following

gamma irradiation as well as during Fenton reactions
[24,25]. X ray crystallographic and NMR studies of
DNA containing 5-HMU show it to assume normal Bform geometry when paired with A [26]. Although the
global conformation of the duplexes is normal and no
distortion is observed, the pucker at the C1 position of
the 2-deoxyribose is changed to exo from the normal
endo pucker [27]. Despite this, 5-HMU pairs correctly in
vitro and is a poor premutagenic lesion [28]. DNA polymerases are not blocked by 5-fU; however, misincorporation of guanine [29] and cytosine [30] opposite the
lesion is observed. When placed into a plasmid vector
and assayed in E. coli, 5-fU causes targeted T 3 C
transitions and T 3 A transversions [31].
DNA containing thymine ring fragmentation, contraction, or ring-open products of thymine have been used as
a template for DNA polymerases. Urea, a fragmentation
product of oxidized thymine, like sites of base loss, lacks
coding and stacking. NMR analysis of a duplex dodecamer containing urea opposite thymine shows that both
are stacked into the helix [32]. In vitro studies with DNA
polymerases have shown that urea is a poorly coding and
stacking lesion and a strong block to DNA synthesis
[13]. In keeping with this observation, urea is also lethal
[18]. When urea is bypassed, purines are the preferred
inserted bases in vitro [21] and mutational studies of urea
in SOS-induced E. coli show that guanine is the major
residue inserted [33]. The ring-open product of dihydrothymine, -ureidoisobutyric acid [21,34], and thymidine
C5-hydrate [34] are also strong blocks to DNA polymerases. Purines and T are inserted opposite the DHT
ring-open product when bypassed in vitro [21] and T 3
A transversions predominate in SOS-induced E. coli
[33]. The phosphoramidite of 5-hydroxy-5-methylhydantoin (hydantoin) was recently synthesized, site specifically incorporated into an oligonucleotide and used as a
template for DNA polymerases [35]. With the three
polymerases tested, E. coli DNA polymerase I Klenow
fragment, Taq DNA polymerase, and Pol , hydantoin
was a strong blocking lesion and with Kf and Taq, dAMP
was the preferred inserted base.
REPAIR OF THYMINE LESIONS

Removal of free radical-damaged thymine products is

initiated by a DNA glycosylase, the first enzyme in the
base excision repair pathway (see [36,37] for reviews).
In vitro, the thymine ring saturation products, the ring
fragmentation, contraction, and ring-open products of
DNA thymine are recognized and removed by the products of the E. coli nth gene (endonuclease III)[38 48],
the nei gene (endonuclease VIII) [49,50], and to a much
lesser extent, the fpg gene (formamidopyrimidine DNA

Consequences of oxidized DNA bases

glycosylase, also called MutM) [35,45,46,51], although

hydantoin and the ring-open form of thymidine C5-hydrate
are efficiently removed by Fpg [35,46]. The ring fragmentation products can also be directly recognized by several
apurinic (AP) endonucleases [52], and thymine glycol is a
substrate for the UvrABC nucleotide [53]. 5-fU paired with
A is removed by AlkA [54,55]; mammalian cells contain a
glycosylase that recognizes 5-HMU [56].
E. coli devoid of both Nth and Nei are spontaneous
mutators [57], but none of the mutations observed are
targeted to thymine, which is in keeping with the observation that the majority of free radical-damaged thymine
bases still correctly pair with adenine. A nth nei fpg alkA
quadruple mutant does enhance mutations in a 5-fUcontaining plasmid [58]. Double nth nei mutants, however, are sensitive to the lethal effects of ionizing radiation and hydrogen peroxide [57,59], which also agrees
with the in vitro studies demonstrating that thymine
glycols, and the thymine breakdown products, are distorting, replication blocking, and lethal lesions. The nth
and nei genes are transcribed as the terminal genes in
operons containing apparently unrelated genes [60], but
do not appear to be induced by agents that produce the
damages the enzymes recognize, although they are induced by a shift from anaerobic to aerobic growth [61].
There are two Saccharomyces cerevisiae Nth orthologs that recognize thymine products [62,63], NTG1,
a mitochondrial activity, and NTG2, primarily present in
the nucleus [64]. There are conflicting data in the literature as to the phenotypes of S. cerevisiae devoid of Ntg1
and Ntg2. In one study [65] double mutants appear to be
spontaneous mutators and sensitive to hydrogen peroxide, while another study [66] shows the double mutants
to have no observable phenotype unless in combination
with mutants defective in recombination, nucleotide excision repair, or translesion synthesis. NTG1 is induced
by menadione [67] and repair of thymine glycol in yeast
has been shown to be coupled to transcription [68]. The
Schizosaccharomyces pombe Nth has a similar substrate
specificity to its orthologs in S. cerevisiae [69,70].
Mammalian Nth orthologs from bovine, mouse, and
human have also been cloned and studied [48,7177].
Several studies have reported human NTH1 to localize
both to the nucleus and the mitochondria [75,78], while
another showed hNTH1 [79] to be exclusively sorted to
the nucleus of HeLa cells and to be cell cycle regulated.
hNTH1 transcription does not appear to be induced by
low doses of ionizing radiation (Inoue and Wallace,
unpublished observations).
FREE RADICAL-DAMAGED CYTOSINE BASES

The major product resulting from hydroxyl radical

attack on DNA cytosine is cytosine glycol, which is

highly unstable and readily deaminates to form uracil

glycol or dehydrates to form 5-hydroxycytosine
(5-OHC) [25,80,81]. Uracil glycol can also dehydrate to
form 5-hydroxyuracil (5-OHU). These latter three products are the major stable free radical-damaged cytosine
products. Several studies have shown that uracil glycol,
5-OHC, and 5-OHU are readily bypassed by DNA polymerases [82,83]. The uracil derivatives, uracil glycol and
5-OHU always pair with adenine, and thus are potentially potent premutagenic lesions [82,83]. 5-OHC pairs
with cognate guanine as well as with adenine and to a
much lesser extent, cytosine [82]. Because they are bypassed by DNA polymerases the oxidized cytosines
should not be lethal lesions. Again, the in vitro studies
with DNA polymerases predict the in vivo results. Studies with single-stranded DNA containing a unique 5-hydroxyuracil or uracil glycol show these lesions not to be
cytotoxic and to induce C 3 T transitions at very high
efficiencies [84]. 5-hydroxycytosine, which is also not
cytotoxic, is much less mutagenic than the uracil derivatives giving C 3 T transitions as well as some C 3 G
transversions [84,85].

REPAIR OF CYTOSINE LESIONS

Free radical-damaged cytosines are repaired by base

excision repair and are recognized by the same glycosylases that remove free radical-damaged thymines, in E.
coli Nth and Nei (for reviews see [86,87]). Fpg also
recognizes 5-OHC and 5-OHU in vitro [45]. Double
mutants deficient in both Nth and Nei are strong spontaneous mutators, about 20-fold above background and
all of the observed mutations are C 3 T transitions
[57,88]. This is in keeping with the substrate specificities
of Nth and Nei, which recognize and remove premutagenic cytosine lesions from DNA. A cellular role for Fpg
in the repair of premutagenic oxidized cytosines is not
supported by the mutation spectrum of nth nei fpg triple
mutants where no increase in C 3 T transitions is found
over that observed in nth nei double mutants (Blaisdell
and Wallace, unpublished observations). As described
above, yeast mutants defective in the Nth orthologs that
recognize oxidized cytosines [67] are at best mild mutators [65]. It should be noted that C 3 T transitions are
one of the major mutational events found in the oxidative
mutational spectra.

CONSEQUENCES OF FREE RADICAL-DAMAGED

PYRIMIDINES

Table 1 summarizes the in vitro and in vivo properties

of representative free radical-damaged thymines and cytosines, and Fig. 1 gives their structures. Of the thymine

S. S. WALLACE
Table 1. Consequences of Representative Free Radical Damaged Pyrimidinesa

Lesion
Thymine glycol
Dihydrothymine
5-Hydroxymethyluracil
5-Formyluracil
Urea
-Ureidoisobutyric acid
Hydantoin
Uracil glycol
5-Hydroxycytosine
5-Hydroxyuracil
Dihydrouracil

Block to DNA
polymerases

Lethal

Base inserted opposite

in vitro

Mutagenic

Yes
No
No
No
Yes
Yes
Yes
No
No
No
No

Yes
No
No
No
Yes
Yes
n.d.
No
No
No
n.d.

AG
A
A
AGC
AG
AGT
AG
A
GA
A
A

Poor
No
Poor
T3C, T3A
T3C
T3A
n.d.
C3T
C3T, C3G
C3T
n.d.

Referenced in text.
n.d. not determined.

moieties that retain intact ring structure, only 5-fU mispairs in vitro and in vivo. When spontaneous mutagenesis is assessed in E. coli lacking the oxidative DNA
glycosylases that recognize free radical-damaged thymines, no mutations at thymine sites are observed. In fact,
mutants devoid of the three oxidative DNA glycosylases,
nth, nei fpg, do not show any mutations at T (Blaisdell
and Wallace; unpublished), even though FPG can also
recognize thymine lesions in vitro. Quadruple mutants,
nth nei fpg alkA, do show an increase in mutations in a
plasmid containing 5-fU in keeping with the in vitro
observation that 5-fU is a substrate for AlkA. See Table
2 for a list of the oxidative DNA glycosylases and their
principal substrates.
When bypassed, purines, principally A, are inserted opposite the thymine ring fragmentation, ring open, and contraction products, most often producing a nonmutagenic
pair. Also, the rate of production of the thymine breakdown products during normal metabolism is considered to
be very low. It is possible that the breakdown products of
thymine might play an as yet undetermined role in spontaneous mutagenesis because in addition to being recognized
by the above-mentioned DNA glycosylases, urea is recognized by the AP endonucleases. Taken together, however,
these data support the idea that oxidized thymines do not
play an important role in mutation induction. On the other
hand, thymine glycol and the thymine breakdown products
are lethal. This is supported by studies with nth nei double
mutants showing them to be hypersensitive to agents that
produce these lesions in DNA. In contrast to free radical
damaged thymine, the cytosine derivatives that retain their
ring structure are all readily bypassed, miscode, and are
potent premutagenic lesions. This is supported by the observation that E. coli mutants devoid of the activities that
recognize oxidized cytosines are mutators with all of the
mutations targeted at cytosine.
In eukaryotes redundant systems apparently play an
important role in processing oxidized pyrimidines. For

example, in S. cerevisiae, mutants defective in the pyrimidine-specific DNA glycosylases do not have strong
phenotype [66]. It remains to be determined what role the
oxidized pyrimidine lesions play in mutagenesis and
carcinogenesis in mammalian systems.
FREE RADICAL-DAMAGED GUANINE BASES

The eighth position on the imidazoyl ring of guanine is

the most readily oxidized leading to the formation of 7,8dihydro-8-oxoguanine (8-oxoG). 8-oxoG has been found in
DNA following treatment with various oxidizing agents,
metals, and ionizing radiation, and is commonly used as a
biomarker of oxidative stress [89,90]. The 8-oxoG adduct
has been extensively structurally characterized both by
NMR and x-ray crystallography. When paired with cytosine, the overall duplex structure is very similar to an
unmodified duplex with the 8-oxoguanine, assuming an
anti confirmation and appropriately base paired to cytosine
[91,92]. Analysis of 8-oxoG opposite adenine shows the
8-oxoG to assume a syn conformation forming a stable
Hoogsteen base pair with adenine in the anti alignment
[93,94]. These structural observations provide the framework for both biochemical studies with DNA polymerases
and the mutagenic analysis of DNA containing 8-oxoG.
Like the oxidized cytosines studied thus far, 8-oxoguanine is readily bypassed by all DNA polymerases
tested [9598]. Depending on which polymerase is used,
there is preferential insertion of cognate C or the noncognate A. Thus the in vitro data predict that because of
its ability to mispair with A, 8-oxoguanine would not be
a lethal lesion but a mutagenic lesion. Interestingly, the
replicative polymerases tested showed significant incorporation of dATP opposite 8-oxoG, while the repair
polymerases preferentially insert dCTP [96].
In keeping with its ability to mispair with A in vitro, an
8-oxoG residue site specifically introduced into a plasmid
vector and replicated in E. coli gives rise exclusively to G

Consequences of oxidized DNA bases

Fig. 1. Structures of the representative pyrimidine base lesions described in Table 1.

3 T transversions at the site of the adduct [95,97,98]. In

HeLa cells a site-specific 8-oxoG adduct in a doublestranded shuttle vector also induced targeted G 3 T transversions [99]. Similar results were found with the singlestranded shuttle vector in COS cells [100]. Of interest, a
sequence context surrounding 8-oxoG that reduced Fpg
cleavage and promoted mispairing with A in vitro was
recapitulated surrounding G 3 T transversions in bacterial
and human oxidative DNA databases [101].
Ionizing radiation also produces the imidazoyl ring frag-

mentation product of guanine 2,6-diamino-4-hydroxy-5formamidopyrimidine (Fapy-G). Although the Fapy-G

product has not been synthesized, methyl Fapy-G has been
examined in vitro and shown to be a strong block to DNA
polymerases [102], and a lethal lesion [103].
REPAIR OF GUANINE LESIONS

Protection of E. coli cells from the potential mutagenic effects of 8-oxoG is multifaceted [104]. E. coli

S. S. WALLACE
Table 2. Oxidative DNA Glycosylases and their Preferred Natural
Substrates

Glycosylase
Nth (endo III) orthologs
MutY orthologs
Ogg orthologs
Fpg orthologs
Nei (endo VIII) orthologs

Principal substrate
Oxidized pyrimidines, Fapy-G
A 8-oxoG
8-oxoG C
8-oxoG C
Oxidized pyrimidines, Fapy-G

formamidopyrimidine DNA glycosylase (Fpg, MutM)

efficiently removes 8-oxoG from DNA when paired with
C. If this repair does not take place prior to DNA replication, A may be inserted opposite the 8-oxoG. To
counteract this potentially mutagenic event, the MutY
protein removes the A mispaired with 8-oxoG. To further
avoid any potential mutations from 8-oxoG adducts, the
MutT protein hydrolyzes 8-oxodGTP in the nucleotide
pools to 8-oxodGMP preventing it from being incorporated opposite A during DNA replication. E. coli cells
defective in Fpg are not sensitive to oxidizing agents but
are mild mutators [105,106]. Addition of the mutY mutation to fpg mutants confers a strong spontaneous mutator phenotype, about 400-fold over that of wild type
[106]. Biologically active DNA exposed extracellularly
to ionizing radiation also shows an increase in G 3 T
transversions in fpg and fpg mutY mutants [107109].
Interestingly, addition of the nei mutation (endo VIII) to
the fpg mutY double mutant confers an additional 3-fold
increase in spontaneous mutation frequency [88]. Single
nei mutants have no observable phenotype and when the
nei mutation is combined with an nth mutation, all the
spontaneous mutations are at cytosines [88]. However, in
the absence of Fpg and MutY, Nei must be able to
remove some potentially premutagenic 8-oxoG lesions
from DNA. In vitro, endo VIII does have activity against
8-oxoG paired with C, albeit weak [88]. (Others have
observed Nei activity on 8-oxoG paired with A [110], but
this in vitro observation is not consistent with the biological result that in the absence of MutY, Fpg and Nei,
all the mutations are G 3 T transversions.) Methyl
Fapy-G is removed by Fpg [111,112], Ogg1, the eukaryotic functional homolog of Fpg [113115], and is a
substrate for the Nth orthologs and Nei [116].
In E. coli, fpg and mutY are transcribed as part of
operons containing genes apparently unrelated to repair
of oxidative damage [117]. Using RNAase protection,
fpg and mutY transcripts were shown not to be induced
by DNA-damaging agents [61], but are increased after
shift from anaerobic to aerobic growth [61,118]. Fpg
activity does appear to be downregulated by metal chelators [119]; however, no differences in fpg and mutY
transcript levels are found in arcA, fnr and fur mutants
[61].

Fpg is found across the bacterial kingdom but is

absent in the Archaea and Eukaryota, with the exception
of the yeast Candida albicans and the plant Arabidopsis
thaliana [120,121]. In eukaryotic cells, the primary activity that removes 8-oxoG from DNA is not an ortholog
of Fpg protein, but rather a member of the Nth superfamily. This protein, called Ogg1, is found across the
eukaryotic kingdom and has a similar substrate specificity to Fpg, that is, it preferentially removes 8-oxoG
opposite C as well as the Fapy products [113115,122].
Like Fpg in E. coli, Ogg in Saccharomyces cerevisiae is
not essential [123]. S. cerevisiae Ogg1 mutants are not
sensitive to the oxidizing agent hydrogen peroxide, but
they do exhibit a mutator phenotype and accumulate G
3 T transversions [123]. Interestingly the yeast S. cerevisiae does not possess an ortholog of the MutY protein,
and 8-oxoG-A mispairs formed during replication are
processed by mismatch repair [124]. However, a MutY
ortholog is present in the yeast Schizosaccharomyces
pombe.
Interestingly, in Drosophila melanogaster, the ribosomal protein S3 has both DNA glycosylase activity
against 8-oxoG as well as an AP lyase activity [125]. S3
also catalyzes the release of Fapy-G from gamma-irradiated DNA [126] and expression of S3 protein in E. coli
fpg mutants suppresses the spontaneous mutator phenotype [125]. Drosophila also have a functional Ogg protein [127].
Orthologs of the yeast Ogg1 are also found in mammalian cells [128 136]. -hOGG1 appears to localize to
the nucleus while -hOGG1 localizes to mitochondria
[78]. Ogg1 activity has been shown to be induced in rat
kidneys by potassium bromate, a renal oxidative carcinogen [137], by peroxisome proliferators [138], crocidolite asbestos [139], and upon administration of diesel
exhaust particles [140]. Increased Ogg activity has also
been demonstrated in ischemic reperfused mouse brain
[141] and rat hearts [142], hOGG1 transcripts are not
induced by low doses of ionizing radiation (Inoue and
Wallace, unpublished observations) and expression of
hOGG1 does not appear to vary during cell cycle [143].
These data suggest that OGG is a housekeeping enzyme
that is induced under some conditions of oxidative stress.
The hOGG1 gene is localized to chromosome 3p26.2
[132], a frequent site of loss of heterozygosity in many
types of human tumors, including clear cell carcinomas
of the kidney [144], head and neck cancers [145], and
small cell lung carcinomas [146,147]. In carcinomas of
the kidney, loss of heterozygosity for the Ogg gene
occurs in 85% of the cases examined [144]. A number of
mutations are also found in the hOGG1 gene of tumor
cells [144,147]. The most common variant amino acid
substitution is Ser326Cys, which does not alter the activity of hOGG1 [148]. Rarer polymorphisms including

Consequences of oxidized DNA bases

substitution of glutamine for arginine at position 46 and

histidine for arginine at position 154 do lead to hOGG1
activity that is less efficient and selective than that of
wild type [149]. Interestingly, a leukemic cell line,
KG-1, has a homozygous Arg209Glu mutation that renders hOGG1 nonfunctional [150]. Mice nullizygous for
Ogg1 exhibit only a mild spontaneous mutator phenotype in certain tissues [151,152], which is similar to the
mutator phenotype observed in ogg mutant yeast and fpg
mutant bacteria.
Orthologs of the MutY protein MYH [153155] are
found in mammalian cells. Human MYH is sorted to the
nucleus and mitochondria as determined by alternative
splicing [156 158]. However, the mice nullizygous for
Myh and both OGG1 and MYH also appear to have no
significant phenotype [159], suggesting that alternative
routes for repairing this potent premutagenic lesion exist
in mammalian cells.
FREE RADICAL-DAMAGED ADENINE BASES

The 8 position on the imidazoyl ring of adenine is also

susceptible to oxidation to 7,8-dihydro-8-oxoadenine (8oxoA). 8-oxoA has been observed in DNA treated by
ionizing radiation and other oxidizing agents [160 162].
Structural studies show that an 8-oxoA paired with T
causes little disturbance of a duplex nonomer and remains in the anti position [163]. In crystals, 8-oxoA
assumes the syn position paired with G anti [164]. In
vitro studies with DNA polymerase I Klenow exo and
Taq DNA polymerase show that 8-oxoA is not a blocking lesion and that thymine is exclusively incorporated
opposite 8-oxoA [163]. In keeping with these observations with bacterial polymerases, 8-oxoA site specifically
incorporated into a phage genome and replicated in E.
coli cells is not cytotoxic or mutagenic [165]. However
with mammalian DNA polymerase , some dGTP is
incorporated opposite 8-oxoA and pol can incorporate
both dGTP and dATP [166,167]. Single 8-oxoA lesions
have been shown to cause A 3 G and A 3 C mutations
in mammalian cells [168].
2-Hydroxyadenine (2-OHA) is observed in DNA of
cells oxidized by metals or ionizing radiation [169 172].
Primer extension studies using templates containing
2-OHA show it to be bypassed and all bases to be
misincorporated [173,174]. With single- and doublestranded vectors containing a single 2-OHA lesion and
replicated in E. coli no cytotoxicity is observed, but A 3
T and A 3 G substitutions are found [175]. With an
SV40 vector containing a single 2-OHA transfected into
COS-7 cells, a targeted 1 deletion was the most frequent mutation observed followed by A 3 G transitions
[176].
4,6-Diamino-5-formamidopyrimidine (Fapy-A) is

readily produced by oxidizing agents and ionizing radiation [177179]. Fapy-A blocks DNA synthesis in vitro
by T7 DNA polymerase, the Klenow fragment of DNA
pol I and polymerase from calf thymus, and thus is a
potentially lethal lesion [180]. -Deoxyadenosine (dA) is a major adenine lesion produced by ionizing
radiation [181] and is a moderate block to DNA synthesis in vitro [182] and in vivo [183]. Targeted mutations
are all one base deletions [183], suggesting that -dA
does not remain stacked in the helix.

REPAIR OF ADENINE LESIONS

8-oxoA is a relatively poor substrate for Fpg [184]

and endo VIII (Dizdaroglu and Wallace unpublished
observations), however, it is efficiently removed by S.
cerevisiae Ogg1 when paired with C [185], which is not
the biological substrate. It may not be important to have
efficient repair of 8-oxoA since it is readily bypassed by
polymerases and most often pairs with T; it is not a lethal
lesion or mutagenic lesion in E. coli and only a weak
mutagenic lesion in mammalian cells.
Little is known about the repair of 2-OHA although
activity against this lesion was found in rat organs [186].
Also, hMYH removes 2-OHA paired with G [157].
Fapy-A, a cytotoxic lesion, is recognized by Fpg protein
and presumably by OGG1 and some orthologs of Nth.

CONSEQUENCES OF FREE
RADICAL-DAMAGED PURINES

Table 3 summarizes the properties of the most wellstudied free radical-damaged purines and Fig. 2 gives
their structures. Of these, 8-oxoG has been the most
frequently studied and is an important premutagenic lesion causing G 3 T transversions. Both E. coli and S.
cerevisiae mutants devoid of the enzymes that process
8-oxoG are strong spontaneous mutators. OGG1 that
removes 8-oxoG is induced in mammalian cells in response to a number of oxidative stressors. Also polymorphisms and loss of heterozygosity of hOGG1 have been
observed in a number of human tumors. However, mice
lacking the glycosylases that process 8-oxoguanine do
not appear to exhibit a major phenotype, although they
do accumulate small numbers of 8-oxoG in the DNA of
certain tissues. With the exception of -adenine, the
oxidized adenines that retain their ring structure primarily pair with cognate T, although the 2-oxoadenine has
been shown to be mutagenic. Both Fapy-G and Fapy-A
are polymerase-blocking lesions; Fapy-G is lethal but not
mutagenic.

S. S. WALLACE
Table 3. Consequences of Free Radical-Damaged Purinesa

Lesion

Block to DNA
polymerases

Lethal

Base inserted opposite

in vitro

Mutagenic

No
Yes
No
No
Yes
Yes

No
Yes
No
No
Yes
n.d.

CA
n.d.
TG
TA
Deletion
n.d.

G3T
No
Poor
A3T, A3G
1 deletion
n.d.

8-Oxoguanine
Fapy-G
8-Oxoadenine
2-Oxoadenine
-Adenine
Fapy-A
a

Referenced in text.
n.d. not determined.

FREE RADICAL DAMAGES OCCURING IN CLUSTERS

In contrast to the free radical damage produced in DNA

from the byproducts of oxidative metabolism, which are
distributed along the DNA molecule, ionizing radiation
produces a burst of hydroxyl radicals during the radiolysis
of water, that can form damage clusters in both DNA
strands [187,188]. When DNA molecules containing
closely opposed clustered base damages are used as substrates for the oxidative DNA glycosylases that recognize
them, [189 193], cleavage of the first strand containing two
opposed damages, is relatively unaffected by the position of
the opposing lesion and a single strand break is formed by
the enzyme that recognizes the damage in the first strand.
However, once a strand break is introduced into one strand
by a DNA glycosylase, cleavage of the damage in the
second strand depends on the position of the opposing
strand break. The excision of the second strand usually
occurs if the opposing strand break is more than one base
away. If excision of both strands does take place, a double
strand break results. In a completely reconstituted base
excision repair system [191] repair of individual strands

usually does not happen, rather a double strand break is

formed depending on whether the DNA glycosylase can
cleave the damage-containing strand closely opposed to the
strand break. These biochemical studies predicted that cells
devoid of the oxidative DNA glycosylases would be radioresistant, whereas cells overproducing an oxidative DNA
glycosylase would be radiosensitive. This was verified by
showing that E. coli triple mutant cells devoid of Nth, Nei,
and Fpg are about twice as radioresistant as wild-type cells
and that this increase in radioresistance could be attributed
to the reduced formation of double strand breaks [194].
Furthermore, the production of the additional double strand
breaks in wild-type cells occurs postirradiation, presumably
due to incisions at closely opposed base damages by the
oxidative DNA glycosylases. Also, cells overproducing
Fpg are radiosensitive and this increase in radiosensitivity is
correlated with an increased formation of double strand
breaks postirradiation. Thus abortive base excision repair
processing of closely opposed free radical-damaged DNA
bases can lead to the formation of lethal double strand
breaks.

Fig. 2. Structures of the representative purine base lesions described in Table 3. -Adenine, not shown, is the anomer of
deoxyadenosine.

Consequences of oxidized DNA bases

CONCLUSIONS

Study of the biological consequences of individual

free radical DNA lesions has been greatly facilitated by
their chemical synthesis and incorporation into DNA
molecules for use as enzyme substrates or as templates
for DNA polymerases. For the lesions studied so far, the
in vitro studies have predicted the cellular results which,
in turn, have been substantiated by studies with DNA
repair defective mutants.

[16]

Acknowledgements Work from the authors laboratory is supported

by NIH R37 CA 33657, R01 CA52040, awarded by the National
Cancer Institute, R01 AG17101-01 awarded by the National Institute
on Aging, and DE-FG02-99ER62861 awarded by the U.S. Department
of Energy. The author wishes to thank past and present students,
postdoctoral associates, and research staff members.

[20]

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