©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping of Copper/Hydrogen Peroxide-induced DNA Damage at Nucleotide Resolution in Human Genomic DNA by Ligation-mediated Polymerase Chain Reaction (*)

(Received for publication, February 10, 1995; and in revised form, May 25, 1995)

Henry Rodriguez (1), Regen Drouin (2)(§)(¶), Gerald P. Holmquist (2), Timothy R. O'Connor (2) (3), Serge Boiteux (3), Jacques Laval (3), James H. Doroshow (1), Steven A. Akman (1)(**)

From the  (1)Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center and the (2)Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, California 91010 and the (3)Institut Gustave Roussy, URA147 CNRS, 94805 Villejuif Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/HO 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)-HO complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-HO complexes and/or the efficiency of base oxidation during resolution of this complex.


INTRODUCTION

Reactive oxygen species (ROS)()induce several classes of DNA damage, including single strand breaks, double strand breaks, modified bases, abasic sites, and DNAprotein 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) .

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/HO-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)-HO 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 HO 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) .


Figure 1: Reactions leading to DNA damage in aerobic solutions. The DNA association constant of Cu(I) is 10(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 HO. 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/HO-induced DNA damage along the PGK 1 gene of male human fibroblast DNA. Our results demonstrate that copper/HO-induced base damage in genomic DNA is nonrandom, exhibiting marked local sequence dependence.


EXPERIMENTAL PROCEDURES

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/HO Treatment

This treatment has been described in detail elsewhere. 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 HO 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 HO were replaced by HO. 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 Endonuclease V

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 NaHPO, 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.

For all enzymes, digestions were stopped by adding 250 µl of HO 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.

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.

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).

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.



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 HO, 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 HO. 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.

The [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.


RESULTS

Excision of Damaged Bases from Plasmid-subcloned P-Labeled PGK 1 Diluted with Genomic DNA Is Sequence-independent

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.

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, 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.


Figure 2: Analysis of sequence context dependence of digestion by Nth or Fpg protein. A, plasmid PGK was end-labeled with 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.


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 (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 HO concentration was determined previously. Treatment of DNA with 50 µM Cu(II), 100 µM ascorbate, and 5 mM HO 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.


Figure 4: Frequency of strand breaks, abasic sites, and modified bases induced by Cu(II)/ascorbate/HO. Purified human male fibroblast DNA was either treated with buffer alone (lanes 2-5) or 50 µM Cu(II), 100 µM ascorbate, 5 mM HO, 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/HO 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/HO-treated DNA (Table 2). The ratio of Cu(II)/ascorbate/HO-induced endonuclease-sensitive base lesions to Cu(II)/ascorbate/HO-induced breaks was 2.7/0.8 = 3.4, high enough for the Cu(II)/ascorbate/HO-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/HO.

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/HO 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/HO-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/HO-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.


Figure 5: A, LMPCR analysis of Cu(II)/ascorbate/HO-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 HO, followed by digestion with Nth plus Fpg proteins. Lanes 8 and 9, LMPCR of Cu(II)/ascorbate/HO-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).


Figure 6: Composite damage map of Cu(II)/ascorbate/HO-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.




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.




DISCUSSION

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)/HO in the supF gene of plasmid pZ189 was limited to sites of polydeoxyguanidylate. Polydeoxyguanidylate was a hot spot for Cu(II)/ascorbate/HO-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)/HO 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)/HO 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.

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 HO causes ``site-specific'' DNA damage. The site-specific nature of DNA base damage induced by copper ion-mediated reduction of HO 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)-HO 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 HO.

Copper ion/ascorbate/HO 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.

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) 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)-HO complex or the efficiency of base oxidation by this complex are important determinants of the damage distribution.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant CA-53115 from the National Cancer Institute (to S. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Unite de Recherche en Genetique Humaine et Moleculaire, Centre de Recherche, Hopital St. Francois d'Assise, Quebec, Canada G1L 3L5.

Holds a Centennial Fellowship from the Medical Research Council of Canada.

**
To whom correspondence and reprint requests should be addressed: Dept. of Cancer Biology, Comprehensive Cancer Center of Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 910-716-0230; Fax: 910-716-5687.

The abbreviations used are: ROS, reactive oxygen species; LMPCR, ligation-mediated polymerase chain reaction; kb, kilobase(s).

R. Drouin, H. Rodriguez, S.-W. Gao, Z. Gebreyes, T. R. O'Connor, G. P. Holmquist, and S. A. Akman, submitted for publication.


ACKNOWLEDGEMENTS

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 endonuclease V, and Steve Bates for providing cultured fibroblasts.


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