DNA Repair Excision Nuclease Attacks Undamaged DNA
A POTENTIAL SOURCE OF SPONTANEOUS MUTATIONS*
Mark E.
Branum,
Joyce T.
Reardon, and
Aziz
Sancar
From the Department of Biochemistry and Biophysics,
University of North Carolina School of Medicine, Chapel Hill, North
Carolina 27599
Received for publication, February 2, 2001, and in revised form, May 11, 2001
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ABSTRACT |
Nucleotide excision repair is a general repair
system that eliminates many dissimilar lesions from DNA. In an effort
to understand substrate determinants of this repair system, we tested
DNAs with minor backbone modifications using the ultrasensitive
excision assay. We found that a phosphorothioate and a
methylphosphonate were excised with low efficiency. Surprisingly, we
also found that fragments of 23-28 nucleotides and of 12-13
nucleotides characteristic of human and Escherichia coli
excision repair, respectively, were removed from undamaged DNA at a
significant rate. Considering the relative abundance of undamaged DNA
in comparison to damaged DNA in the course of the life of an organism,
we conclude that, in general, excision from and resynthesis of
undamaged DNA may exceed the excision and resynthesis caused by DNA
damage. As resynthesis is invariably associated with mutations, we
propose that gratuitous repair may be an important source of
spontaneous mutations.
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INTRODUCTION |
Nucleotide excision repair is a general repair system that removes
damaged bases from DNA by dual incisions of the damaged strand at some
distance from the lesion, releasing the damaged base in the form of
12-13-mers in prokaryotes and 24-32-mers in eukaryotes (1, 2). It is
the major repair system for bulky base adducts, but it also acts on
nonbulky lesions such as oxidized or methylated bases and, as such,
functions as a backup system for DNA glycosylases, which have
restricted substrate ranges (3, 4).
The wide substrate spectrum of the excision nuclease raises two
interrelated questions: what is the substrate range of the enzyme
system and how does the enzyme recognize substrate? Both of these
questions have been addressed in numerous studies, and at present we
have a basic understanding of damage recognition in both prokaryotes
and eukaryotes (1, 2, 5, 6). With regard to substrate range, its limits
remain to be defined. The excision nuclease, which originally was
thought to be specific for bulky lesions, was later found to excise
nonbulky adducts such as methylated bases but, apparently, failed to
excise nucleotides with backbone modifications such as the C4' pivaloyl
adduct (5, 6). With the availability of more efficient in
vitro systems (4, 7, 8) we decided to re-examine the question of
recognition of backbone modifications. We found that both
phosphorothioate and methylphosphonate backbone modifications were
recognized as substrates by the human excision nuclease. This, in turn,
led us to take a closer look at the effect of the enzyme system on undamaged DNA. We find that both the human and the Escherichia coli excision nucleases excise oligomers of 23-28 and 12-13
nucleotides, respectively, from undamaged DNA. This gratuitous excision
and the inevitable repair synthesis that must follow could be potential sources of spontaneous mutations. Our data suggest that even in nondividing cells in which there is no DNA replication, there can be
significant DNA turnover due to gratuitous excision and resynthesis and
that this gratuitous "repair" may cause mutations in such cells,
even when they are protected from all extrinsic and intrinsic DNA
damaging agents.
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MATERIALS AND METHODS |
Substrates--
Linear DNA substrates (136 or 140 bp1 in length) were prepared
with centrally located lesions as described previously (3). Unmodified
oligonucleotides were obtained from Operon Technologies (Alameda, CA).
The methylphosphonate-containing 12-mer, the
phosphorothioate-containing 12-mer, and the 8-hydroxyguanine-containing
(8-oxoG) 11-mer were purchased from Midland Scientific Reagent Company
(Midland, TX). The sequence of the centrally located 12-mers was
5'-GAAGCTACGAGC with the phosphorothioate or methylphosphonate
modifications between C5 and T6. The oligomer (5'-GTA[TT]ATG)
containing the (6-4) photoproduct was prepared and high performance
liquid chromatography-purified as described previously
(9) and used to assemble a 136-bp substrate.
Repair Factors--
Cell-free extracts (CFE, 10-20 mg/ml) were
prepared as described previously (4) and stored at
80 °C in
storage buffer (25 mM HEPES-KOH (pH 7.9), 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM dithiothreitol, and 12.5%
(v/v) glycerol). The Chinese hamster ovary cell lines were obtained
from the American Type Culture Collection (Manassas, VA): CRL 1859 (AA8, wild type), CRL 1860 (UV41, XP-F mutant), and CRL 1867 (UV135,
XP-G mutant). XPF·ERCC1 was purified using a previously described
chromatographic scheme after expression in an insect cell system (8).
The UvrABC proteins were purified as described elsewhere (10).
Excision Assay--
In vitro removal of
oligonucleotides was measured with the excision assay, which measures
the release of radiolabeled fragments from substrate DNA (11). For
experiments with the mammalian excision nuclease, the reaction mixtures
contained 3 fmol of radiolabeled substrate DNA, 12.6 fmol of pBR322,
and 50 µg of CFE in 25 µl of reaction buffer (17 mM
HEPES-KOH (pH 7.9), 12 mM Tris-HCl (pH 7.5), 35 mM KCl, 44 mM NaCl, 5.8 mM
MgCl2, 0.3 mM EDTA, 0.34 mM dithiothreitol, 2-4 mM ATP (except where indicated
otherwise), and 2.5% glycerol with bovine serum albumin at 200 µg/ml) and were incubated at 30 °C for 60 min. For complementation
assays, 25 µg of each repair-deficient CFE was premixed on ice and
used in the reaction, or 20 ng of XPF·ERCC1 was added to 50 µg of
XP-F-deficient CFE. For experiments with the E. coli
excision nuclease, the reactions contained 3 fmol of radiolabeled
substrate DNA, 5 nM UvrA, 20 nM UvrB, and 50 nM UvrC in 25 µl of reaction buffer (50 mM
Tris (pH 7.5), 50 mM KCl, 10 mM
MgCl2, and 2 mM ATP with bovine serum albumin
at 100 µg/ml) and were incubated at 30 °C for 60 min. Following
the reaction, the DNA was extracted with phenol:chloroform, and the
deproteinized DNA was precipitated with ethanol, resuspended in
formamide/dye mixture, and resolved in 10% polyacrylamide gels containing 7 M urea (sequencing gels) to separate excision
products from substrate DNA. DNA was visualized by autoradiography or
by scanning on a model 860 Storm PhosphorImager (Molecular Dynamics), and the intensity of signal was analyzed with ImageQuant software (version 5.0, Molecular Dynamics). The extent of excision for each
reaction was determined from the percentage of signal migrating as
~23-28-mers (12-13-mers for E. coli excinuclease)
relative to the signal for full-length DNA (signal in the
~110-140-mer range, which contains 80-90% of the total
radioactivity in the lane). Because of the significant DNA degradation
observed with CFE-based reactions, we adjusted for nonspecific nuclease
activity by determining the percentage of signal in an equal sized area that migrated in the ~30-38-mer range and subtracting this
background value from the percentage excision calculated for fragments
migrating in the ~23-28-mer range.
Assay for Cryptic Oxidative DNA Damage--
Oligonucleotides,
either as purchased from the supplier (Operon Technologies) or after
being subjected to mock kinase and ligase reactions followed by
purification via denaturing polyacrylamide gel electrophoresis and
annealing, were hydrolyzed with formic acid; the hydrolysates were
lyophilized, trimethylsilylated, and analyzed for 8-oxoG by isotope
dilution mass spectrometry as described previously (12). The analysis
was kindly performed by Dr. Miral Dizdaroglu (National Institute of
Standards and Technology, Gaithersburg, MD).
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RESULTS |
Excision of DNA Backbone Modifications--
To define the
substrate range of human excision nuclease, a variety of lesions have
been tested. Fig. 1 shows some of the substrates that we have used in this and our previous studies. Prior
work has shown that all base lesions and even mismatches tested were
excised by the human excision nuclease, albeit with greatly different
efficiencies (3, 4). However, attempts to detect excision from DNA with
backbone modifications failed, suggesting that these lesions might not
be substrates for the human excision nuclease (5, 6). Recently, we have
improved the efficiency and sensitivity of the excision assay (13, 14), and we wished to test DNA with backbone modifications in our assay system. Fig. 2 shows that DNA containing
either a phosphorothioate or a methylphosphonate in the backbone are
recognized and excised by the human excision nuclease, suggesting that
not only base modifications but also backbone modifications, which
cause modest helical distortions, can be substrates for the excision
nuclease.

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Fig. 1.
Structures of DNA modifications incorporated
into excision repair substrates. A, the
phosphorothioate (S)- and methylphosphonate
(ME)-adducted thymidines introduce minor alterations into
the sugar-phosphate backbone and are shown next to undamaged
(unmodified, UM) thymidine. Thymine glycol (Tg)
and 8-oxoguanine (8-oxoG) are damaged bases generated by
reactive oxygen species; these lesions cause minor helical distortions.
The lower panel illustrates three bulky, helix-distorting
lesions introduced either by ultraviolet radiation, such as cyclobutane
thymine dimer (T<>T) and (6-4) photoproduct
(T[6-4]T), or by the chemotherapeutic agent cisplatin,
1,2-d(GpG) diadduct (cis-Pt). Only unmodified,
phosphorothioate, methylphosphonate, and T[6-4]T substrates were used
in the current study; the other substrates have been tested previously
(1). B, schematic illustration of the substrates. The duplex
of 136/140 bp without lesion or with a lesion at the position indicated
by a circle and a radiolabel at the position indicated by an
asterisk was prepared as described previously (3).
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Fig. 2.
Excision of oligonucleotides containing DNA
backbone modifications by mammalian excision nuclease. Substrate
DNAs were incubated for 60 min in 25-µl reaction mixtures containing
50 µg of AA8 CFE plus 3 fmol of DNA substrate and then resolved in a
10% polyacrylamide sequencing gel; brackets indicate the
location of excision products. Excision products were only observed in
complete reactions (i.e. those containing both CFE and ATP,
lanes 2, 5, and 8). The observed
percentages of excision (n = 3-4 experiments) were
0.31 ± 0.1 for phosphorothioate (S), 0.43 ± 0.21 for methylphosphonate (ME), and 12.8 ± 3.5 for
T[6-4]T. Only of the reactions were loaded onto the lanes
containing the T[6-4]T substrate.
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Repair of Undamaged DNA by Human Excision Nuclease--
The
difference between the backbones of unmodified DNA with phosphodiester
and phosphorothioate linkages is minor (15). Hence, the excision of an
oligomer carrying the phosphorothioate bond was unexpected and led us
to consider the possibility that the excision nuclease system, which is
capable of recognizing such subtle perturbations in the duplex
structure, may act in a similar manner on unmodified DNA with a low but
finite probability. When we tested the unmodified DNA in our assay
system, we found that fragments 23-28 nucleotides in length
were removed from this substrate as well (Fig.
3). That these oligomers are generated by
the excision nuclease system and not by nonspecific degradation of DNA
by contaminating nucleases is supported by three lines of evidence.
First, the excision nuclease is the only known mammalian nuclease that
cuts out oligomers in the range of 23-28 nucleotides from a duplex.
Second, the excision of 23-28-mers is ATP-dependent as is
the excision nuclease in removing damaged bases. Finally, extracts from
cells lacking the XPG or XPF subunits of the excinuclease fail to
release 23-28-mers from undamaged DNA. Moreover, the excision activity
can be restored by supplementing extract from the mutant cell line with
the missing subunit. In conclusion, the data in Fig. 3, considered in
its entirety, show that the human excision nuclease is capable of
excising oligomers of 27 nt nominal length from undamaged DNA. Similar
levels of excision were observed when the centrally located oligomer
with 32P label in the 140-143-bp duplex was an undamaged
15-mer 5'-TCCTCCTCGCCTCCT or 20-mer, 5'-GCTCGAGCTAAATTCGTCAG (data not
shown). Thus, it appears that excision of undamaged DNA occurs in at
least three different sequence contexts.

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Fig. 3.
Excision of undamaged DNA by the mammalian
excision nuclease. Substrate DNA prepared with unmodified
oligonucleotides was incubated for 60 min in 25-µl reaction mixtures
either lacking or containing cell-free extracts prepared from normal
cells (AA8), repair-deficient cell lines (XPF or
XPG), or XPF extract supplemented with purified protein or
with extract prepared from an XPG cell line. The figure shows an
autoradiograph obtained after resolution of DNA samples in a 10%
polyacrylamide sequencing gel; brackets indicate the
location of excision products. Excision products were not observed in
the absence of cell extracts (lane 1) or when substrate was
incubated with extracts prepared from repair-deficient cell lines
lacking excision nuclease subunits (lanes 3 and
6); but the defect in XPF extracts was restored to wild type
levels (lane 2) by the addition of recombinant XPF·ERCC1
(F-E1) heterodimer (lane 4) or by coincubation
with the XPG cell extract (lane 5). The observed percentages
of excision were 0.09, 0.07, and 0.11, respectively, for AA8, XPF
extract complemented with recombinant protein and XPF extract mixed
with XPG cell extract; the apparent presence of excision products in
lanes 1, 3, and 6 were not above
background levels. In separate experiments (data not shown,
n = 3) conducted under the same conditions, the
percentages of excision were 0.07 ± 0.02 for undamaged DNA and
10.4 ± 2.3 for T[6-4]T photoproduct when substrates were
incubated with AA8 CFE.
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Repair of Undamaged DNA by E. coli Excinuclease--
The subunits
of human and E. coli excinucleases do not share any
homology, yet the reaction mechanisms of the two systems are remarkably
similar (1): ATP-independent damage recognition followed by
ATP-dependent unwinding of DNA and formation of a stable
preincision complex and finally dual incisions at phosphodiester bonds
several base pairs removed from the damage site. Hence, the finding
that the human excision nuclease performs standard dual incisions on
backbone modified and undamaged DNA led us to re-examine the effect of
E. coli excinuclease on these substrates as well. The
results, presented in Fig. 4, show that
it does excise a characteristic 12-nucleotide oligomer from DNA with
phosphorothioate or methylphosphonate modifications. This excision is
also observed with undamaged DNA, albeit at lower efficiency. As with
the human excision nuclease, excision was also observed in a 140-bp
duplex when the centrally located oligomer was a 20-mer,
5'-GCTCGAGCTAAATTCGTCAG (data not shown). Thus, it appears that removal
of oligomers of defined lengths from damaged or undamaged DNA is a
general property of excision nucleases.

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Fig. 4.
Excision of oligonucleotides containing
undamaged DNA and backbone modifications by the E. coli
excision nuclease. Undamaged (UM) and
phosphorothioate (S)- and methylphosphonate
(ME)-adducted thymidines were incorporated into DNA
substrates, and the (6-4) photoproduct (T[6-4]T) was used
as a reference lesion. Substrate DNA (3 fmol) was incubated for 60 min
in 25-µl reaction mixtures containing 5 nM UvrA, 20 nM UvrB, and 50 nM UvrC in reaction buffer with
and without ATP as indicated. The figure shows an autoradiograph
obtained after resolution of DNA samples in a 10% polyacrylamide
sequencing gel; brackets indicate the location of excision
products. Note that the major excision products from undamaged and
T[6-4]T substrates are 12 nt in length. The photoproduct causes one
nucleotide slower migration than expected. The phosphorothioate and
methylphosphonate modifications may also cause slightly anomalous
migration of the oligomer. Excision products were only observed in
complete reactions (i.e. those containing both UvrABC and
ATP, lanes 2, 5, 8, 11),
and the observed percentages of excision were 0.68 ± 0.4 (n = 7) for undamaged, 0.94 ± 0.6 (n = 6) for phosphorothioate, 1.23 ± 0.5 (n = 2) for methylphosphonate, and 50.6 ± 12.5 (n = 5) for T[6-4]T substrates.
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Damage in "Undamaged DNA"--
Although we have interpreted
the excision from our DNA oligomers prepared without targeted lesions
as arising from undamaged DNA, it is virtually impossible to have a DNA
preparation free of damage (16-18). This is because both the
nucleobases and the phosphodiester bonds are rather reactive and prone
to modification by both extrinsic and intrinsic agents. Thus, a given
DNA preparation always contains a certain amount of lesions, the level
of which would be dependent on a variety of factors, including the
source of DNA and the method of purification. In particular, it is
practically impossible to prepare DNA without oxidative base damage.
Hence, it could be argued that the excision we observe from our
nominally undamaged DNA may be due to low levels of oxidative damage
introduced during the handling of DNA.
To address this concern we measured the level of 8-oxoG in our
synthetic oligonucleotides that had been subjected to essentially the
same treatment as our radiolabeled substrate. 8-OxoG is the most common
oxidative stress lesion (16, 18) and, among the major oxidative base
lesions, it is the most efficient substrate for human excision nuclease
(4). Thus, we reasoned that if excision from undamaged DNA arises from
cryptic lesions, most of it would have been caused by 8-oxoG. The rate
of excision from undamaged DNA is 5-10% the rate of removal of a
single 8-oxoG in the same duplex (Ref. 4 and data not shown). Hence, if
excision from the undamaged DNA were to arise from cryptic 8-oxoGs in
our substrate, it would be expected that the 9 guanines, which are close enough to the radiolabel to give rise to radiolabeled 23-28-mer products, would be in the form of 8-oxoG in 5-10% of the undamaged DNA. This would mean an 8-oxoG/G ratio in the undamaged DNA substrate of (0.05 to 0.10)/9 = 5.5 × 10
3 to
1.1 × 10
2. As shown in Table
I, 8-oxoG is present at a level of
5.2 × 10
4 to 5.4 × 10
4 in our DNA. Hence cryptic 8-oxoG
contributes in the range of 5-10% to the excision signal from our
undamaged DNA. Thymine glycol, urea, and other oxidative lesions, which
are less frequent than 8-oxoG (16, 18), and are excised less
efficiently by the human excision nuclease (4), are expected to
contribute to the signal from undamaged DNA even less. It should be
noted, however, that our quantitation of 8-oxoG was performed with
nonradiolabeled DNA. An argument could therefore be made that with
radiolabeled DNA the 8-oxoG level would be greater due to DNA damage
caused by radioactive decay, and thus there would be higher
contributions to the excision signal from damage. However, we think
this is unlikely to be the source of gratuitous excision for the
following reason. The DNA molecules in which the decay occurs are no
longer detectable in the excision assay, and the likelihood that low level
-decay would damage other DNA molecules, especially in the
presence of EDTA in the storage buffer, is infinitesimally small. Thus,
it can be reasonably concluded that most of the excision signal we
detect with undamaged DNA is produced by the attack of the excision
nuclease on undamaged DNA as a consequence of the intrinsic property of
the action mechanism of the excision nuclease system.
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DISCUSSION |
Our findings raise two questions: why and how is the undamaged DNA
attacked by the excision nuclease, and what is the biological role of
gratuitous DNA repair? These questions are addressed below.
Attack of Excision Nuclease on Undamaged DNA--
The precise
mechanism of damage recognition by human excision nuclease is not
known. Hence, at present, it is not possible to answer the questions of
why and how the enzyme attacks undamaged DNA in any detail. Based on
the structure of a preincision DNA-enzyme complex, which contains a
subset of the repair factors and partially unwound and kinked DNA (1,
2, 7, 13), it has been proposed that any DNA structure that is amenable
to unwinding and kinking and otherwise assuming the conformation
existing in the ultimate preincision complex might function as a
substrate (1, 5, 6). Since even undamaged DNA can assume the
conformation of the preincision complex with low but finite probability
(1), it is not surprising that undamaged DNA is a substrate for
excision nucleases. Indeed, there have been reports on
incision of undamaged DNA by the E. coli excinuclease (19,
20). In one of those studies, however, uniformly radiolabeled plasmid
DNA was used in a nicking assay that is incapable of detecting an
excinuclease mode of action (19). The second study used linear DNA
uniformly labeled with 32P as a substrate in an excision
assay, and 9-nt-long oligomers were released instead of the
characteristic 12-13-nt-long oligomers (20). Later work revealed that
the 9-mers are released by a potent 3'-exonuclease action of the UvrABC
proteins at a nick or a double-strand break (21, 22), and hence the
product was not released by an excinuclease type of action (dual
incisions in one strand). In this study we present unambiguous evidence that both the human and the E. coli excision nucleases
attack undamaged DNA in the typical excinuclease mode.
It is very likely that certain DNA sequences would be more susceptible
to attack by excision nuclease than others. We have tested three
different random sequences and found a similar level of excision by the
excision nuclease. A more extensive survey, however, is likely to
identify certain sequences and conformations with increased
susceptibility to excision nuclease. Indeed, a recent study (23) has
shown that the poly(purine:pyrimidine) tract present in the polycystic
kidney disease gene (PKD1) when present in a
supercoiled plasmid is efficiently processed by the E. coli
excinuclease. An extreme case of the effect of DNA conformation on
gratuitous repair is the form of gratuitous repair that has been
proposed to occur as a side product of transcription-repair coupling
(24, 25). It has been speculated that when RNA polymerase stalls at
natural transcriptional pause sites the transcription-coupled repair
machinery is activated in a manner similar to RNA polymerase stalling
at a lesion and that such activation of the transcription-coupled repair system leads to gratuitous and potentially mutagenic repair. Currently there is no experimental evidence for gratuitous repair initiated by stalled RNA polymerase. However, there are several reports
that show that transcribed DNA is mutated at higher frequency than
nontranscribed DNA (26-29). Whether this increased mutation frequency
is due to transcription-coupled gratuitous DNA repair or the increased
susceptibility of single-stranded DNA in the transcription bubble to
various DNA damaging agents is not known at present.
Biological Relevance of Gratuitous Repair--
We suspect that
gratuitous excision repair has no beneficial effect for the organism.
Removal and replacement of undamaged DNA by nucleotide excision repair
is the price the cell has to pay to have an omnipotent DNA repair
enzyme capable of handling a virtually infinite variety of lesions.
This excision and resynthesis may not be totally innocuous, since it
may introduce spontaneous mutations into undamaged DNA as is shown in
the following calculation.
Fig. 5 compares the relative efficiency
of human excision nuclease on a variety of lesions and on undamaged
DNA. As is apparent, with the unique substrate and assay system we use,
undamaged DNA is excised at a rate of about 1% that of the (6-4)
photoproduct, which is the best natural substrate for the enzyme and is
used as the "gold standard" for other substrates. However, in
calculating the susceptibility of undamaged DNA to excision nuclease
activity with the (6-4) photoproduct as a reference, a correction
factor must be introduced for the relative abundance of the targets. Essentially all of the excision products from the (6-4) substrate arise
from a single lesion, whereas the excision products from undamaged DNA
arise from dual incisions over about a 50-nucleotide region in a
variety of combinations that bracket the radiolabel (Fig.
6). Hence, in calculating the efficiency
of the enzyme on an undamaged nucleotide, a correction factor of 50 is
introduced, making the actual efficiency of an undamaged base relative
to that of a (6-4) photoproduct equal to about 1/(50 × 100) = 2 × 10
4. This might seem
insignificant, but if one considers that every nucleotide in the human
genome complement is a potential target for attack by the excision
nuclease, the level of excision of undamaged DNA becomes significant.
The maximum rate of excision of (6-4) photoproducts under substrate
saturating condition has been estimated to be 2.7 × 103/min/diploid human cell (30). Assuming that the relative
rates we obtained in vitro are applicable to the in
vivo environment, it is predicted that every minute (2.7 × 103) × (2 × 10
4) = 5.4 × 10
1 undamaged nucleotides would be subject to
excinuclease action, and since each excision event removes about 25 nucleotides, it is calculated that 5.4 × 10
1 × 25 = 13.5 nucleotides/min are
removed by the human excision nuclease. This, in turn, means excision
and replacement of about 2 × 104 nucleotides per day
per human cell. This value is comparable with the nucleotide turnover
that occurs under physiological conditions as a result of base excision
repair processing of damaged bases (104 to 105
per cell per day) arising from depurination, deamination, oxidation, and methylation (31, 32). Thus, it is conceivable that gratuitous nucleotide excision repair contributes to DNA turnover as much as base
excision repairs acting on spontaneous DNA lesions. Gratuitous repair
is not necessarily restricted to the nucleotide excision repair system.
It has been shown that certain DNA glycosylases also attack undamaged
DNA causing gratuitous repair which, under special conditions, can be
mutagenic (33, 34). Mismatch repair, like nucleotide excision repair,
has a wide substrate range and many mechanistic similarities to
nucleotide excision repair (35, 36) and conceivably may perform
gratuitous repair. Since the mismatch repair patches, as a rule, are
much larger than those of base or nucleotide excision repair, this
system as well may contribute to spontaneous mutagenesis.

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Fig. 5.
Relative levels of oligonucleotide excision
by the mammalian system. To express the relative efficiency of
nucleotide excision for various DNA substrates, the average percent
excision observed for the T[6-4]T photoproduct was defined as 100, and values for the excision of other lesions were normalized relative
to this value. For comparison, all excision reactions were under
substrate-limiting conditions, and the sources of excision nuclease
were cell-free extracts prepared from mammalian cells. This figure
incorporates data generated in the present study and in previously
published work where (6-4) photoproduct or cyclobutane thymine dimer
were used as references (4, 37); abbreviations are the same as in Fig.
1. Note that the spacing of the lesions along the x axis is
for the sake of clarity and is not meant to imply a special
relationship between the various structures and their efficiency as
substrates.
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Fig. 6.
Dual incisions releasing labeled
oligonucleotides from undamaged and damaged DNA. The (6-4)
photoproduct is released mainly by incisions at the 4th
phosphodiester bond 3' and the 24th phosphodiester bond 5'
to the lesion. With undamaged DNA any combination of incisions about 28 nt apart, bracketing the label, release the appropriate size fragments.
The dual incisions representing the extreme locations for releasing the
label are shown, and any combination of sites between these two
extremes will release the radiolabel from undamaged DNA.
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Conclusion--
In this paper we have shown that DNA with minor
backbone modifications and nominally unmodified (undamaged) DNA are
attacked by the human and E. coli excision nucleases. The
concern that the nominally undamaged DNA may in fact contain some
cryptic damage can never be unequivocally eliminated. We feel, however,
that the excision we observe from undamaged DNA does represent attack on truly undamaged DNA for the following reasons. First, using an
analytical probe for the most common spontaneous lesion in DNA, 8-oxoG,
we demonstrate that the level of this lesion in our synthetic substrate
is well below the level required to account for the level of excision
we observe for such undamaged substrate. Second, the fact that even
such a minor modification as the replacement of an oxygen by a sulfur
in the backbone increases the susceptibility of DNA to the excision
nuclease leads to the reasonably logical conclusion that substrate and
nonsubstrate DNA are not quantized for the excision nuclease. Instead
it suggests that DNA structures ranging from gross distortions to no
distortion represent the two extremes of the continuum of excision
nuclease substrates.
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ACKNOWLEDGEMENTS |
We thank Deborah Croteau and Vilhelm Bohr for
critical comments on the manuscript. We are grateful to Miral
Dizdaroglu for determining the background 8-oxoG levels in our
undamaged DNA substrates.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM32833.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Biophysics, Mary Ellen Jones Bldg., CB#7260, University of North
Carolina School of Medicine, Chapel Hill, NC 27599-7260. Tel.:
919-962-0115; Fax: 919-843-8627; E-mail:
Aziz_Sancar@med.unc.edu.
Published, JBC Papers in Press, May 15, 2001, DOI 10.1074/jbc.M101032200
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ABBREVIATIONS |
The abbreviations used are:
bp, base pair(s);
CFE, cell-free extract;
XP, xeroderma pigmentosum;
ERCC, excision repair cross-complementing;
8-oxoG, 8-hydroxyguanine;
nt, nucleotide(s).
 |
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