(Received for publication, February 10, 1995; and in revised form, May 25, 1995)
From the
The ligation-mediated polymerase chain reaction was used to map
the frequency of reactive oxygen species-induced DNA damage at
nucleotide resolution in genomic DNA purified from cultured human male
fibroblasts. Damaged pyrimidine and purine bases were recognized and
cleaved by the Nth and Fpg proteins from Escherichia coli,
respectively. Strand breaks and modified bases were induced in
vitro by copper ion-mediated reduction of hydrogen peroxide in the
presence of ascorbate; reactant concentrations were adjusted to induce
lesions at a frequency of 1 per 2-3 kilobases in purified genomic
DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand
breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively
to remove adventitious transition metal ions. Ligation-mediated
polymerase chain reaction analysis of the damage frequency in the
promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that
Cu(II)/ascorbate/H
Reactive oxygen species (ROS)
The
transition metal ion-catalyzed reduction of hydrogen peroxide has
served as a useful model reaction for generating ROS. DNA base damage
caused by reduction of hydrogen peroxide by Fe(II) or Cu(I) has been
quantified in vitro(18, 19) and in vivo(20) by analytic techniques, principally gas
chromatography-mass spectrometry (18, 19, 20) or high performance liquid
chromatography with electrochemical
detection(21, 22, 23) . Such techniques have
revealed the types and amounts of modified bases induced by
ROS(18, 19, 20) , but not their distribution
along DNA sequences.
Cu(II)/ascorbate/H
Figure 1:
Reactions leading to DNA damage in
aerobic solutions. The DNA association constant of Cu(I) is
10
For all enzymes, digestions were stopped by adding 250
µl of H
The
enzyme-treated samples were phenol/chloroform-extracted and
ethanol-precipitated. All samples were separated by electrophoresis
through 8% polyacrylamide-7 M urea gels, followed by
autoradiography.
About 500 base pairs, on both strands, of the human PGK 1 gene, including the promoter region and the first exon, were
studied using the primer sets described in Table 1. DNA purified
from HeLa cells was treated with base-specific chemical cleaving agents (38) . Chemically cleaved G, G + A, T + C, and C
samples were run along with the other samples through LMPCR and were
included on the sequencing gels to provide base position markers.
The
[
The rate of
endonuclease-dependent cleavage of damaged base positions may depend on
the local sequence. If true, this would produce misleading LMPCR lesion
frequency patterns that would vary with the degree of endonuclease
digestion. Consequently, we developed terminal endonuclease digestion
conditions so that incision occurred at all cleavable adducts
independent of sequence-related rate differences. To determine
conditions in which digestion is terminal and sequence-independent, a
plasmid bearing the PGK 1 promoter region and first exon,
Figure 2:
Analysis of sequence context dependence of
digestion by Nth or Fpg protein. A, plasmid PGK was
end-labeled with
Figure 3:
Analysis of glycosylase kinetics by
neutral glyoxal gel electrophoresis. A, purified human male
fibroblast genomic DNA was treated with either 0 (lanes 1 and 2) or 1.5 mM KMnO
Figure 4:
Frequency of strand breaks, abasic sites,
and modified bases induced by
Cu(II)/ascorbate/H
Figure 5:
A, LMPCR analysis of
Cu(II)/ascorbate/H
Figure 6:
Composite damage map of
Cu(II)/ascorbate/H
Figure 7:
The LMPCR-derived damage intensities at
each position in Fig. 6were analyzed for sequence dependence.
Five hot spot motifs were found; the number of + signs above each
position corresponds to the average damage intensity at positions along
such motifs.
The LMPCR technique has sufficient resolution to map several
types of DNA base damage in human genomic DNA (35, 39, 42, 55, 56) and
was applied here to map ROS-induced base damage. With the exceptions of
the unsaturated products 5-hydroxymethyl-2`-deoxyuridine and
5,6-dihydroxy-2`-deoxycytidine, the major species of ROS-induced base
modifications are well suited for mapping by LMPCR, because they are
substrates for cleavage either by Nth protein (modified pyrimidines) (46, 47) or Fpg protein (modified
purines)(48, 49) . The associated lyase activity of
these enzymes cleaves the phosphodiester backbone to produce the
5`-phosphoryl groups that are substrates for LMPCR.
The ability of
LMPCR to faithfully reflect the actual damage distribution induced by a
flux of ROS is critically dependent on complete cleavage by damage
recognition/incision enzymes at each position. Our control experiments
confirm that this criterion is fulfilled by Nth and Fpg proteins for
certain lesions in the PGK 1 promoter region. However, if
specific lesions are cleaved at a much lower rate than those examined
in our control experiments, their presence might not be observed by
LMPCR. Also, other regions of the genome may impose sequence-dependent
restrictions on cleavage of modified bases not imposed by the PGK 1 promoter.
Mapping of the copper/hydrogen peroxide-induced DNA
damage frequency has, until now, been limited to the study of
piperidine-labile damage in genes cloned into plasmid DNA. Sagripanti
and Kraemer (33) observed that piperidine-labile DNA damage
induced by Cu(II)/H
Kinetic(24, 25, 26, 27) ,
inhibitor(18, 19, 25) , and copper
ion-mediated DNA cleavage (28, 29) studies have
suggested that the oxidizing species produced by reaction of DNA-Cu(I)
complexes with H
Copper
ion/ascorbate/H
In summary,
we have shown that LMPCR is a useful technique for mapping ROS-induced
DNA base modifications at nucleotide resolution in target genes in
genomic DNA. Its utility is enhanced by using the damage-specific
enzymes Nth protein and Fpg protein as cleaving agents, which provide
improved efficiency and specificity compared with chemical cleaving
agents. The distribution of copper/hydrogen peroxide-induced base
modifications in the promoter and first exon of PGK 1 in human
genomic DNA is nonrandom and sequence-dependent; the 5` bases of
d(pG
We thank Dr. Richard Cunningham (State University of
New York) for gifts of Nth protein and endonuclease IV, Dr. Steven
Lloyd (University of Texas Medical Branch at Galveston) for supplying
the T
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O
caused DNA base damage by a
sequence-dependent mechanism, with the 5` bases of
d(pG
) and d(pC
) being damage
hot spots, as were the most internal guanines of d(pGGGCCC) and
d(pCCCGGG). Since base damage occurs after formation of a
DNA-Cu(I)-H
O
complex, these data suggest that
the local DNA sequence affects formation of
DNA-Cu(I)-H
O
complexes and/or the efficiency of
base oxidation during resolution of this complex.
(
)induce
several classes of DNA damage, including single strand breaks, double
strand breaks, modified bases, abasic sites, and DNA
protein
cross-links (reviewed in (1, 2, 3) ). Such
damage is of potential pathobiologic significance, because many
ROS-induced base modifications are
promutagenic(4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
ROS-induced DNA damage has been linked to cancer and
aging(14, 15, 16, 17) .
O
-mediated DNA damage in
aerobic aqueous solutions is believed to be induced in vitro and in vivo through formation (Fig. 1) of a
DNA-Cu(I)-H
O
complex(24, 25, 26) . Kinetic
analysis(24, 25, 26, 27) , inhibitor
studies(18, 19, 25) , and studies of copper
ion-mediated cleavage of small radiolabeled DNA molecules (28, 29) suggest that the reaction of Cu(I)-DNA
complexes with H
O
results in the induction of site-specific oxidative DNA damage and oxidation of the Cu(I)
by mechanisms still in
dispute(25, 27, 30, 31, 32) .
(58) , whereas that of Cu(II) is
10
(6) . Consequently, Cu(II) distributes almost
equally between DNA bound and free solution forms. In the presence of
an excess of ascorbate, soluble Cu(II) is reduced and tightly bound to
DNA; bound Cu(II) may also be reduced. The DNA-Cu(I) then forms a
complex with H
O
. During the resolution of this
complex, DNA damage occurs by mechanisms still in
dispute(30, 43, 45, 63) , and
DNA-bound Cu(I) is oxidized.
To date, mapping of copper/HO
-induced DNA
base damage at nucleotide-level resolution has been limited to
piperidine-sensitive damage in small target genes subcloned
into plasmids(31, 33, 34) , and the identity
of the modified bases that facilitated the piperidine cleavage has not
been determined. The ligation-mediated polymerase chain reaction
(LMPCR) is an extremely sensitive technique for mapping the frequency
of rare DNA breaks along genes at nucleotide
resolution(35, 36, 37, 38, 39) .
We have used LMPCR in conjunction with Nth protein (also designated
endonuclease III) and Fpg protein (also designated formamidopyrimidine
glycosylase), enzymes which recognize and cleave DNA at oxidized bases,
to map in vitro copper/H
O
-induced DNA
damage along the PGK 1 gene of male human fibroblast DNA. Our
results demonstrate that copper/H
O
-induced base
damage in genomic DNA is nonrandom, exhibiting marked local sequence
dependence.
DNA Preparation
Human male skin fibroblasts were
grown in 150-mm dishes to confluent monolayers in
Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. Cells were harvested, and DNA was isolated as described
previously (40) .
(
)After
phenol/chloroform extraction, the DNA was precipitated in ethanol,
redissolved in 10 mM HEPES, 1 mM EDTA, pH 7.4, at 70
µg/ml, then dialyzed against distilled water overnight at 4
°C.
Copper/Ascorbate/H
This treatment has been described in detail
elsewhere.O
Treatment
Briefly, 10 µg of dialyzed DNA incubated at
room temperature for 30 min with 50 µM CuCl
.
Following this, Chelex®-treated potassium phosphate, pH 7.5,
ascorbate, and H
O
were added to final
concentrations of 1 mM, 100 µM, and 5
mM, respectively. The final volume was 268 µl. The
reaction proceeded for 30 min at room temperature with gentle rocking.
The reaction was quenched by addition of EDTA to 2 mM,
followed by precipitation of DNA with 0.3 M sodium acetate, pH
7.0, and 70% ethanol. The DNA pellets were air-dried. The ``no
treatment'' samples went through the same steps except that stock
solutions of CuCl
, ascorbate, and H
O
were replaced by H
O. Treated DNA was analyzed for
lesion types and frequency by glyoxal gel electrophoresis as described
elsewhere (Footnote 2, as modified from (41) ).
Enzyme Digestion
For all enzyme digestions,
10-µg aliquots of DNA were digested in a volume of 100 µl, and
all incubations with the enzymes were done at 37 °C for 60 min. The
``no enzyme'' samples were incubated at 37 °C in the Nth
protein buffer.
Nth Protein from Escherichia coli and Fpg Protein of E.
coli
DNA aliquots were digested in 50 mM Tris-HCl, pH
7.6, 100 mM KCl, 1 mM EDTA, pH 8.0, 0.1 mM
dithiothreitol, and 100 µg/ml bovine serum albumin. Enzyme was added in 5 µl of dilution buffer (10% glycerol, 50
mM Tris-HCl, pH 7.6, 100 mM KCl, 1 mM EDTA,
pH 8.0, 0.1 mM dithiothreitol, and 500 µg/ml bovine serum
albumin).
Endonuclease IV (the Nfo Protein) from E. coli
DNA
aliquots were digested in 50 mM HEPES, pH 7.6, 100 mM KCl, 1 mM EDTA, pH 8.0, 1 mM dithiothreitol, and
50 µg/ml bovine serum albumin. Two µl of the stock enzyme
solution (about 1.7 mg/ml in 50% glycerol and 10 mM HEPES)
were added and incubated for 30 min. One additional µl of the
enzyme was added for the last 30 min of the incubation period.
T
DNA aliquots were
digested in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1
mM EDTA, pH 8.0, 1 mM dithiothreitol, and 100
µg/ml bovine serum albumin(42) . Four µl of the stock
enzyme solution (about 150 µg/ml in 25 mM NaH Endonuclease V
PO
, pH 6.8, 100 mM NaCl, and 1
mM EDTA) were added and incubated for 30 min. Another three
µl of the enzyme were added for the last 30 min of the incubation
period.
O and 50 µl of 0.8% SDS and mixing well.
Phenol/phenol-chloroform/chloroform extractions were carried out to
remove the proteins. DNA was precipitated by a 10-min incubation on dry
ice after addition of 18 µl of 5 M NaCl and 1000 µl of
cold 100% ethanol. The air-dried pellet was dissolved either with the
mix for glyoxal gel (extraction not necessary when for gel only) or
Sequenase buffer (40 mM Tris-HCl, pH 7.7, and 50 mM NaCl) at a concentration of 0.16 µg/µl in preparation for
LMPCR.
Nth and Fpg Protein Digestion of Cloned DNA
The 5`
control region and first exon (positions -436 to +371) for
human phosphoglycerate kinase 1 (PGK 1) (43) was
subcloned in Bluescript SK (Stratagene). After
digestion with XbaI, XhoI, and SacI, the
fragments were labeled at the 3` end of the upper strand with Escherichia coli DNA polymerase (Klenow fragment) and
[
P]dCTP. For T + C and G + A samples,
the labeled fragments were mixed with 14 µg of herring sperm
carrier DNA and were subjected to base-specific chemical DNA
sequencing(38) . For KMnO
and methylene blue
treatment, the labeled fragments were mixed with 10 µg of genomic
human DNA. (a) The labeled fragments and genomic DNA were
treated with 1 mM KMnO
in 0.3 M ammonium
chloride, pH 8.6, and allowed to react at room temperature for 15
min(8) . The treated DNA was cleaved with different amounts of
Nth protein. (b) The labeled fragments and genomic DNA were
dissolved in a 200-µl solution containing 10 mM potassium
phosphate, pH 7.6, buffer and 2 mM methylene blue and exposed
to white light (100-watt tungstram 7 Sylvania bulb) in a microtiter
plate at a distance of 17 cm from the light bulb(44) . The
treated DNA was cleaved with different amounts of Fpg protein.
LMPCR
The LMPCR protocols used for this work were
modified from Pfeifer et al.(40) . The procedure can
be divided into six steps: 1) primer extension of an annealed
gene-specific oligonucleotide (primer 1) to generate blunt ends, 2)
ligation of a universal asymmetric double-strand linker, 3) PCR
amplification using a second gene-specific oligonucleotide (primer 2),
4) separation of the DNA fragments on a sequencing polyacrylamide gel,
5) transfer of the DNA to a nylon membrane by electroblotting, 6)
hybridization of a radiolabeled probe prepared by repeated primer
extension using a third gene-specific oligonucleotide (primer 3).
Primer Extension
Primer 1 was extended in
siliconized 0.625-ml tubes; a thermocycler (MJ Research Inc.) was used
for all incubations. DNA (1.28 µg) was diluted in a volume of
15-18 µl of a solution containing 40 mM Tris-HCl, pH
7.7, 50 mM NaCl, and 1 pmol of primer 1. DNA was denatured at
98 °C for 3 min and the primer annealed at 48-50 °C for
15-20 min. After cooling on ice, 9.0 µl of the following mix
were added: 7.5 µl of MgCl-dNTP mix (20 mM MgCl
, 20 mM dithiothreitol, and 0.25 mM of each dNTP), 1.1 µl H
O, and 0.4 µl of
Sequenase 2.0 (13 units/µl, U. S. Biochemical Corp.). The samples
were incubated at 48-49 °C for 5 min, 50 °C for 1 min, 51
°C for 1 min, 52 °C for 1 min, 54 °C for 1 min, 56 °C
for 1 min, 58 °C for 1 min, and 60 °C for 1 min. The samples
were cooled on ice and 6 µl of ice-cold 310 mM Tris-HCl,
pH 7.7, was added. The samples were incubated at 67 °C for 15 min
to inactivate Sequenase, then cooled on ice.
Ligation
The primer-extended molecules that have a
5` phosphate were ligated to an unphosphorylated synthetic asymmetric
double-stranded linker(45) . To each microtube, 45 µl of
the following mix was added: 13.33 mM MgCl, 30
mM dithiothreitol, 1 mM ATP, 83.3 µg/ml bovine
serum albumin, 100 pmol of linker, and 6.25 units of T
DNA
ligase (5 units/µl, Boehringer Mannheim). The samples were
incubated overnight at 18 °C. Then, while kept on ice, 25 µl of
10 M ammonium acetate, 1 µl of 0.5 M EDTA, pH
8.0, 1 µl of 20 µg/µl glycogen, and 260 µl of ice-cold
100% ethanol were added to stop the reaction and precipitate the DNA.
DNA pellets were redissolved in 50 µl of water.
PCR Amplification
50 µl of the Taq polymerase mix (0.02% gelatin, 20 mM Tris-HCl, pH 8.9, 4
mM MgCl, 80 mM KCl, 0.25 mM of
each dNTP, 10 pmol of primer 2, 10 pmol of linker primer (LP25), and
3.0 units of Taq DNA polymerase (5 units/µl, Boehringer
Mannheim) were added to each sample, and the reaction was overlaid with
mineral oil. Reactions underwent 22 PCR cycles of 95 °C for 1 min,
61-73 °C (1-2 °C below T
of primer 2) for 2 min, and 74 °C for 3 min. The
following stop mix was added under the mineral oil layer: 13 µl of
3 M sodium acetate, pH 5.2, 3 µl of 0.5 M EDTA,
pH 8.0, and 9 µl of H
O. The samples were extracted with
250 µl of premixed phenol:chloroform (92 µl:158 µl), then
ethanol-precipitated. Air-dried DNA pellets were dissolved in 7.0
µl of premixed formamide dye (1 part water, 2 parts 94% formamide,
2 mM EDTA, pH 7.7, 0.05% xylene cyanole, 0.05% bromphenol
blue) (38) in preparation for sequencing gel electrophoresis.
Gel Electrophoresis, Electroblotting, and
Hybridization
Half (3.5 µl) of each DNA sample was
electrophoresed through 60-cm 8% polyacrylamide-7 M urea gels
at 70 watts constant power. The DNA was transferred to a charged nylon
membrane (Qiabrane, Qiagen, Chatsworth, CA) by electroblotting using an
HEP3 electroblotting apparatus (Owl Scientific Inc.) according to the
manufacturer's instructions. Blotted DNA was UV-cross-linked
(1200 joules/m) to the membranes.
P]dCTP-labeled single-stranded probe was
prepared by repeated linear primer extension, between 30 and 35 cycles,
by Taq polymerase with primer 3 (see Table 1) on a
double-stranded template of the PGK 1 gene cloned in
Bluescript(43) . Primer 3 is downstream from primer 2 to
increase specificity. To avoid long probes, PGK plasmid was cut with
different restriction enzymes: ApaI for A3, C3, J3, and O3; AluI for E3; EcoRI for D3; BssHII for G3 and
F3; DdeI for H3; and HaeIII for N3. The 150-µl
mix consists of: 0.01% gelatin, 2 mM MgCl
, 10
mM Tris-HCl, pH 8.9, 40 mM KCl, 250 µM of dATP, dGTP, and dTTP, 40 ng of template, 75 pmol of primer 3,
2.5 units of Taq polymerase, and 10 µl of
[
P]dCTP (3000 Ci/mmol). The probe was
precipitated with 37.5 µl of 10 M ammonium acetate, 20
µg of glycogen, and 420 µl of ice-cold 100% ethanol,
resuspended in TE buffer (TE, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), then added to the hybridization tube containing the
prehybridized membrane in 5-6 ml of hybridization solution. After
overnight hybridization at 62-68 °C (2 °C below the
calculated T
of the probe), the membrane
was washed as described(38) .
Autoradiograms
Air-dried membranes were exposed to
Kodak XAR-5 x-ray films for 0.5-8 h with intensifying screens at
-70 °C. On the final autoradiogram, each band represents a
nucleotide position where a break was induced, and the signal intensity
of the band reflects the number of DNA molecules with ligatable ends
terminating at that position. The intensity of the bands was evaluated
visually and confirmed by PhosphorImager scans. The intensity of each
band was plotted on a scale from ``0'' (no band), to
``5'' (the most intense band) after normalization to the
corresponding Maxam-Gilbert sequence lane, in order to allow for
nonlinearity of the primer extension and linker ligation steps.
Excision of Damaged Bases from Plasmid-subcloned
The prerequisite to LMPCR, cleavage
which leaves terminal 5`-phosphoryl groups at the sites of oxidative
base damage, can be accomplished chemically, e.g. by treating
damaged DNA with hot piperidine(31, 33) . However,
chemical cleavage produces a high background of nonspecifically cleaved
nucleotide positions and limits sensitivity to extremely high mutagen
doses(37) . Two enzymes responsible for the excision of a
variety of modified DNA bases induced by ROS are the Nth and Fpg
proteins of E. coli. The Nth protein cleaves the N-glycosidic bond of most oxidatively damaged pyrimidine
bases(46, 47) , whereas Fpg protein similarly cleaves
the N-glycosidic bond of most oxidatively damaged purine
bases(48, 49) . Both of these enzymes possess a lyase
activity that subsequently cleaves at all abasic sites, leaving
terminal 5`-phosphoryl groups, the substrate for DNA ligase whose
frequency distribution is mapped by LMPCR.
P-Labeled PGK 1 Diluted with Genomic DNA Is
Sequence-independent
P-labeled at its 3` end, was treated with KMnO
or methylene blue-white light. KMnO
treatment induces
pyrimidine damage, primarily thymine glycol(8) , which is
cleaved by Nth protein. White light-activated methylene blue induces
modified purines, primarily 8-oxoguanine(44) , which are
cleaved by Fpg protein. The treated plasmids were linearized, diluted
to single copy level with human genomic DNA, and then 10 µg of DNA
was cleaved by either the Nth or Fpg proteins followed by sequencing
gel autoradiography. Bands along the PGK 1 promoter region, as
generated by cleavage with either endonuclease, increased in intensity
with increasing enzyme added over a range of 0-500 ng for Nth
protein (Fig. 2A) or 0-250 ng for Fpg protein (Fig. 2B); no pattern change, and no further cleavage
at any position was achieved by addition of >500 ng of either
protein. These data suggest that detection and cleavage of ROS-modified
bases by these two enzymes at high enzyme concentrations, terminal
digestion conditions, is independent of DNA sequence and represents
100% cleavage of every potentially cleavable position.
P. Sequencing standards were treated with
either formic acid (lane 1) or hydrazine (lane 2).
Experimental samples were treated with 0 (lane 11) or 1.5
mM KMnO
(lanes 3-10).
KMnO
-treated DNA was then digested with 0-750 ng of
Nth protein. All DNA was electrophoresed through an 8% acrylamide-7 M urea gel at a constant power of 70 watts, 50 °C, then
autoradiographed using Kodak XAR-5 film. B,
P-labeled plasmid PGK DNA was treated with either formic
acid (lane 1) or hydrazine (lane 2), 0 (lane
11) or 2 mM methylene blue plus white light (lanes
3-10). Methylene blue-treated (MB) DNA was digested
with 0-500 ng of Fpg protein, then electrophoresed and
autoradiographed.
To further
show that these two endonucleases as used in subsequent LMPCR studies
resulted in terminal digestion, human genomic DNA was treated with
either KMnO or white light-activated methylene blue and
cleaved with various concentrations of Nth and Fpg protein before
glyoxal gel analysis. The size distributions of single-stranded DNA
fragments displayed on the glyoxal gels (Fig. 3) show that
reactions containing 250 ng or more of each enzyme terminally cleaved
10 µg of damaged genomic DNA.
(lanes
4-13), then digested with 0-750 ng of Nth protein.
Digested DNA was analyzed for nick frequency by denaturing neutral
glyoxal gel electrophoresis. Lane 3 is
phage DNA size
markers whose lengths are listed to the left of the
photograph. B, purified human male fibroblast genomic DNA was
treated with either 0 (lanes 1 and 2) or 2 mM methylene blue plus white light (lanes 4-13), then
digested with 0-500 ng of Fpg
protein.
Types and Frequency of Lesions Induced in Genomic DNA by
Copper-mediated Reduction of Hydrogen Peroxide
After
establishing enzyme conditions to terminally digest DNA at sites of
oxidatively damaged bases, we determined the frequency of lesions
induced by copper ion-mediated reduction of HO
.
We used glyoxal gel electrophoresis (Fig. 4) instead of alkaline
electrophoresis to visualize the single stranded M
distribution while avoiding cleavage of alkali-sensitive sites.
In such a gel mobility analysis, M
, the weight
average molecular weight, approximately coincides with the peak density
of mass distribution (50) , and M
, the number average M
, is equal to M
/2. The
density of DNA breaks is
1/M
(37, 50) . The
dependence of induction of breaks and base modification density on
Cu(II) and H
O
concentration was determined
previously.
Treatment of DNA with 50 µM Cu(II), 100 µM ascorbate, and 5 mM H
O
for 30 min at room temperature induced
2.7 glycosylase-sensitive sites/10 kb (Table 2). This is a
sufficient lesion frequency for LMPCR mapping(37) , and these
conditions were used in all subsequent LMPCR analyses.
O
. Purified human male
fibroblast DNA was either treated with buffer alone (lanes
2-5) or 50 µM Cu(II), 100 µM ascorbate, 5 mM H
O
, for 30 min at
37 °C (lanes 6-10). After treatment, DNA was
digested with either buffer alone (lanes 2 and 6),
T
endonuclease V (Endo V) (lanes 3 and 7), endonuclease IV (Endo IV) (lanes 4 and 8), Nth protein (lane 9), or Nth plus Fpg proteins (lanes 5 and 10). Digested DNA was analyzed for
single strand M
by neutral glyoxal gel
electrophoresis. The lesion frequency of each lane is indicated. Lanes 1 and 11 are DNA size markers whose lengths are
shown to the left of the
photograph.
To determine
the frequency of various classes of DNA lesions induced by this
treatment, treated and untreated (incubated in buffer) DNA was digested
with various enzymes (Fig. 4). T endonuclease V and
endonuclease IV both possess activities incising DNA at abasic sites,
but not at ROS-induced base modifications(51, 52) .
Untreated DNA had 0.3 abasic endonuclease-sensitive sites/10 kb above a
0.2 breaks/10 kb background (Table 2).
Cu(II)/ascorbate/H
O
treatment increased the
frequency of single strand breaks to 1 per 10 kb while leaving the
frequency of abasic sites unchanged (Fig. 4, lanes 6 and 7). Nth and Fpg protein both possess an abasic lyase
activity in addition to their pyrimidine and purine glycosylase
activities(51, 53, 54) , so they cleave DNA
at all abasic sites in addition to cleaving sites with oxidative base
damage. After subtracting the contributions of breaks and abasic sites,
Nth and Fpg proteins did not significantly cleave oxidatively damaged
base sites in untreated DNA, but cleaved 2.7 such sites per 10 kb in
Cu(II)/ascorbate/H
O
-treated DNA (Table 2). The ratio of
Cu(II)/ascorbate/H
O
-induced
endonuclease-sensitive base lesions to
Cu(II)/ascorbate/H
O
-induced breaks was 2.7/0.8
= 3.4, high enough for the
Cu(II)/ascorbate/H
O
-induced
endonuclease-sensitive base lesions to be detected above the strand
break signal by LMPCR. There was more 2-fold interexperimental
variation in the base lesion:strand break ratio. In other repetitions
of this experiment, the ratio was as high as 8.0.
The 3.4
value shown here is the minimum base lesion:strand break ratio induced
by Cu(II)/ascorbate/H
O
.
The Distribution of Damage Induced by Copper Ion-mediated
Reduction of Hydrogen Peroxide in Human PGK
1
Cu(II)/ascorbate/HO
-induced base
damage along the promoter and first exon of human PGK 1 in
purified genomic DNA. PGK 1 is a single copy housekeeping gene
located on the X chromosome; male fibroblast DNA was used to study the
transcriptionally active gene. Table 1lists the primer sets used
for LMPCR and their positions relative to the transcription start site
of PGK 1. Fig. 5shows the autoradiographs generated by
LMPCR using primer sets E (mapping the transcribed strand) and F
(mapping the nontranscribed strand). These two primer sets map
complementary strands in the region, including the transcription start
site for PGK 1. Weak signals from strand breaks were apparent
after Cu(II)/ascorbate/H
O
treatment (lanes
8 and 9). The average stoichiometric ratio of base damage
signal intensity to strand break signal intensity determined by
PhosphorImager analysis was 4.3, which was comparable with the 3.4
ratio determined by glyoxal gel analysis. The comparability of the base
damage:strand break ratio determined by the two techniques suggests
that the majority of
Cu(II)/ascorbate/H
O
-induced strand breaks are
detectable by LMPCR, i.e. they have ligatable 5`-phosphoryl
ends. Also, with the exception of a single position representing an
LMPCR artifact (open arrow), untreated DNA contained no
significant signal generated by Nth + Fpg protein digestion (lane 10). This artifact was subsequently obviated by
appropriate temperature ramping during the primer extension step. The
distribution of Cu(II)/ascorbate/H
O
-induced
modified bases detectable by Nth + Fpg protein cleavage was
nonuniform, with hot and cold spots (lanes 5-7). Nth
protein- and Fpg protein-digested DNA were combined so that pyrimidine
and purine base damage could be visualized on the same lane of the
autoradiogram. For purposes of illustration, the sequence of one hot
spot mapped by primer set E and its complement mapped by primer set F,
along with arrows indicating high intensity signals, are shown
in Fig. 5.
O
-induced DNA damage in the
promoter region of human PGK 1 using primer set E (transcribed
strand). Lanes 1-4, LMPCR of DNA treated with standard
Maxam-Gilbert cleavage reactions. Lanes 5-7, LMPCR of
DNA treated with 50 µM Cu(II), 100 µM ascorbate, 5 mM H
O
, followed by
digestion with Nth plus Fpg proteins. Lanes 8 and 9,
LMPCR of Cu(II)/ascorbate/H
O
-treated DNA
digested with buffer only. Lane 10, LMPCR of DNA treated with
buffer only, followed by digestion with Nth plus Fpg proteins. In order
to guide the reader, a small portion of the Maxam-Gilbert-derived
sequence is shown to the left of the autoradiogram. The arrows indicate which positions in this region show Nth or Fpg
protein-cleavable damage. B, LMPCR analysis using primer set F
(nontranscribed strand). All lanes represent the same treatments as
described in A.
Autoradiograms similar to those shown in Fig. 5were generated using all of the primer sets listed in Table 1, covering both strands of the entire promoter region of PGK 1. The damage intensity at each position is illustrated in Fig. 6. Cu(II)/ascorbate/HO
-induced base
modifications were distributed nonuniformly throughout the entire
region. Guanine bases were most frequently modified, followed by
cytosine bases. Thymine bases were modified infrequently and adenine
bases only rarely. Five G-C-rich sequence motifs were especially prone
to base modification (Fig. 7). Characteristic damage patterns
were evident within these motifs. The 5` base of
d(pG
) and d(pC
) were most
often modified, as was the most internal guanine of d(pGGGCCC) or
d(pCCCGGG). Interestingly, in all the sequence motifs containing
polydeoxyguanidylate, alternate guanines were either damage-prone or
damage-resistant (Fig. 7).
O
-induced Nth or Fpg
protein-cleavable DNA base damage in the promoter and first exon of
human PGK 1 genomic DNA. Damage was assessed by LMPCR using
primer sets covering both strands of the entire region shown. Damage
was quantified by visual inspection of autoradiograms such as the two
shown in Fig. 5. Quantitative damage assignments were confirmed
by direct PhosphorImager (Bio-Rad) analysis of radioactive filters;
signal intensities were quantified using RFLPScan software (Billerica,
MA). The height of the bar at each position on the map shown here
corresponds to damage intensity at that position. The bottom strand is
the transcribed strand.
O
in the supF gene
of plasmid pZ189 was limited to sites of polydeoxyguanidylate.
Polydeoxyguanidylate was a hot spot for
Cu(II)/ascorbate/H
O
-induced base modifications
in PGK 1 in genomic DNA as well; however, several other
sequence motifs were identified as hot spots as well. Yamamoto and
Kawanishi (31) observed that piperidine-labile DNA damage
induced by Cu(II)/H
O
in the human c-HA-RAS gene cloned into pUC18 was limited to thymines and guanines.
Thymines and guanines 5` to guanines were especially damage-prone. They
carried out similar experiments with Cu(I)/H
O
and observed damage hot spots at thymines of
d(pGTC)(34) . Modified thymines were rare in PGK 1 in
genomic DNA. Although several d(pGTC) sites were present, they were not
hot spots for modified thymine. It is difficult to compare these
previous studies with ours, because it is unknown which types of
oxidatively modified bases are piperidine-labile (probably a subset of
Nth or Fpg protein-cleavable bases). It is possible that certain
piperidine-labile thymine lesions are not as susceptible to cleavage by
Nth protein, in which case they may have been underrepresented by
LMPCR.
O
causes
``site-specific'' DNA damage. The site-specific nature of DNA
base damage induced by copper ion-mediated reduction of
H
O
initially suggested that the damage
distribution is intimately related to the sequence dependence of copper
ion binding affinities in DNA. The known sequence-dependent aspects of
copper ion binding are limited; poly(dG-dC) sequences have higher
binding affinity than do poly(dA-dT)
sequences(24, 57, 58, 59, 60) .
However, under our reaction conditions the molar ratio of copper ion to
DNA-phosphates was 1:3, a ratio at which all copper ion DNA-binding
sites are saturated(61, 62) . This implies that copper
ion binding is a necessary but insufficient requirement for the
observed base damage pattern; factors in addition to copper binding
affinity are important determinants of the sequence-dependent pattern
of DNA base damage. One such factor is the local efficiency of
formation of the DNA-Cu(I)-H
O
complex, which is
a necessary intermediate in the DNA oxidation
reaction(24, 31, 33) . The local geometry
must be able to accommodate hydrogen peroxide at a coordination site in
the copper complex, probably by displacing water. Another such factor
is the local efficiency of DNA base oxidation by the copper-oxo
complex. Molecular models of Cu(II)-water complexes bound to DNA which
were built using crystal-derived coordinates as initial conditions
suggest that C8 of guanine located 5` to bound copper makes the closest
approach to the ligand water molecules(63) ; C5 and C6 of a
cytosine located 5` to bound copper also closely approach the ligand
water molecules. Since C8 of guanine and C5 and C6 of cytosine are the
principal sites of base oxidation observed with copper/hydrogen
peroxide(18) , the sequence motifs we observed to be
damage-prone may reflect local geometries which most closely
approximate guanine C8 or cytosine C5/C6 and copper-coordinated
H
O
.
O
induces DNA strand breaks in
addition to base modifications. The majority of strand breaks terminate
with a ligatable 5`-phosphoryl, permitting mapping by LMPCR. Strand
breaks occur primarily at deoxyguanidylate and deoxycytidylate sites.
Although the average ratio of strand breaks:base damage is
1:4,
this ratio varied markedly from site to site, which is consistent with
kinetic data, suggesting that strand breaks and modified bases are
produced by different reaction mechanisms.
) and d(pC
), as well as
the most internal deoxyguanidylate of d(pGGGCCC) or d(pCCCGGG), are
damage hot spots. Local factors influencing the efficiency of formation
of the DNA-Cu(I)-H
O
complex or the efficiency
of base oxidation by this complex are important determinants of the
damage distribution.
endonuclease V, and Steve Bates for providing
cultured fibroblasts.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.