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,
,1,
* Department of Pathology and Laboratory, School of Medicine;
Department of Environmental Sciences and Engineering; and
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina, USA
2Correspondence: Department of Pathology and Laboratory, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA. E-mail: pchastai{at}med.unc.edu
ABSTRACT
Reactive oxygen species (ROS) and the oxidative DNA damage they produce [e.g., 8-oxo-guanine and apurinic/apyrimidinic (AP) sites] have been linked to the pathogenesis of several age-related and chronic diseases. The basal number of AP sites measured in DNA by immuno-slot-blot analysis ranges from 70,000 to 100,000 per genome. We used electron microscopy to determine how AP sites were distributed in isolated DNA fibers from fresh calf thymus and HeLa cell cultures. We observed that AP sites were not equally distributed throughout all the fibers. A small percentage of the analyzed DNA fibers contained a disproportionate amount of the total AP sites in nonrandom groups of 10 to >30 closely spaced in a small region (e.g., 20 AP sites in a 6 kb length of DNA). This finding suggests that genomic sites may differ in their vulnerability to ROS damage, perhaps because of local chromatin structure. Nonrandom AP site formation also suggests that the detrimental effects of ROS in the development of disease may be related not simply to the total number of AP sites present but to how AP sites are distributed along a DNA fiber and, perhaps, to the genomic sites affected.—Chastain, II, P. D., Nakamura, J., Swenberg, J., Kaufman, D. Nonrandom AP site distribution in highly proliferative cells.
Key Words: abasic • apurinic/apyrimidinic • electron microscopy • oxidative damage • reactive oxygen species • methoxyamine • isolated DNA fibers
REACTIVE OXYGEN SPECIES (ROS) are a class of reactive ions and free radicals generated within cells by oxidative reactions both as products of endogenous metabolism and in response to environmental exposures. Inside the cell, ROS are generated in a variety of ways: as byproducts of energy production in mitochondria and detoxification reactions by cytochrome P-450 enzymes and in response to microbial agents. Many environmental agents also induce the formation of ROS in cells. Once formed, ROS can react with the DNA creating numerous products of oxidative DNA damage (1)
. It is thought that the base excision repair (BER) pathway repairs these DNA base lesions (in addition to DNA lesions generated by alkylation and deamination). The normal antioxidant defense mechanisms of the cell are able to eliminate most of ROS and prevent formation of ROS-induced apurinic/apyrimidnic (AP) sites, but when the cell cannot eliminate enough ROS, oxidative stress occurs.
Exposure to an excess of reactive oxygen radicals resulting in oxidative stress and cellular damage has been linked to the pathogenesis of certain age-related and chronic diseases, aging, and some forms of cancers (1
, 2)
. The exact mechanism of how oxidative DNA damage increases disease development is currently not known, but the number of oxidative damage products is thought to be a prominent factor. Thus, most research has been done using approaches that detect the overall number of oxidative DNA damage sites.
In using only global types of analysis, the subtle contributions of additional aspects, such as where and how the damage is distributed, have not been considered in any mechanism trying to connect oxidative damage and disease. To determine whether AP sites form clusters, we characterized how oxidative damage in the form of apurinic/apyrimidinic/abasic (AP) sites was distributed in genomic DNA fibers obtained from calf thymus and HeLa cells using electron microscopy (EM).
MATERIALS AND METHODS
Detection of endogenous AP sites by slot blot
DNA was isolated from fresh calf thymus (CT) or HeLa cells using a method that does not introduce any additional AP sites (3
4
5)
. The amount of isolated DNA was determined by reading the absorbance of the DNA solution at 260 nm and then multiplying the value by a factor of 50 to obtain the DNA concentration in µg/ml. The global level of AP sites in genomic DNA was determined using the AP slot blot assay (ASB) method described by Nakamura et al. (5)
. Briefly, 15 µg of DNA in 150 µl of PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaH2PO4·7H2O, and 1.4 mM KH2PO4, pH 7.4) were incubated with 1 mM biotinylated aldehyde reactive probe (ARP; Dojindo Laboratories, Kumamoto, Japan) at 37°C for 10 min. ARP reacts with the aldehydic group of the ring open form of AP sites and thus covalently places a biotin moiety at each AP site. Since the biotin moiety is what is determined in the slot blot assay, any AP sites that may form after the ARP incubation step (i.e., the heat-denaturation step and baking steps discussed below) will not contribute to the number of AP sites detected in the samples.
After precipitation using cold ethanol, ARP-reacted DNA was resuspended in Tris-EDTA (10 mM Tris-HCl and 1 mM EDTA, pH 7.2) buffer. DNA concentration was measured as described above, and a DNA solution was prepared at 1.5 µg in 100 µl of Tris-EDTA buffer. Heat-denatured DNA (by heating to 100°C for 5 min followed by rapid cooling to 4°C) was immobilized on a nitrocellulose membrane using a slot blot apparatus. The membrane was soaked in 5x saline-sodium citrate (SSC; 0.75 M NaCl and 0.075 M trisodium citrate) and baked at 80°C in a vacuum oven for 1–2 h. The membrane was preincubated with 10 ml of 20 mM Tris-HCl buffer (pH 7.5), containing 0.1 M NaCl, 1 mM EDTA, 0.5% casein, 0.25% BSA, and 0.1% Tween 20, and incubated at room temperature for 40 min in the same solution containing streptavidin-conjugated horseradish peroxidase (BioGenix, San Ramon, CA, USA). After being rinsed with washing buffer [20 mM Tris-HCl (pH 7.5), 0.26 M NaCl, 1 mM EDTA, and 0.1% Tween 20], the enzymatic activity on the membrane was visualized by the use of enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA). The image was collected on X-ray film, and the developed film was analyzed using an Ultrascan XL scanning densitometer.
EM detection of induced AP sites in control DNA
Plasmid DNA, 
3,000 bp, containing on average between 20 and 50 AP sites per 100,000 bp (0.6–1.5 per plasmid) as determined by the slot blot assay (data not shown), was incubated with 50 mM methoxyamine for 30 min at 37 C°, and excess methoxyamine was removed by gel filtration. Methoxyamine reacts with the aldehyde group present on carbon 1 (C1') of the deoxyribose after a base loss and thereby protects the deoxyribose group from reaction with ARP (6)
. After the endogenous AP sites had been masked, the plasmid DNA was incubated at 70°C in acid buffer for 100, 200, and 400 s, and the number of new AP sites was determined by slot blot analysis using the method described by Nakamura et al. (5)
and above. Plasmid DNA with the induced AP sites was incubated with 1 mM ARP for 60 min at 37°C in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA (TE). The ARP-treated DNA was filtered through 2-ml columns of Bio-Gel A5M (Bio-Rad, Hercules, CA, USA) equilibrated with 10 mM TE, incubated with 10-nM streptavidin coated gold particles (nanogold, Nanoprobes, Yaphank, NY, USA), and filtered through Biogel A5M once again. The samples were absorbed to thin carbon foils, washed, air-dried, and rotary shadowcast with tungsten at high vacuum (7
, 8)
. Samples were examined in a Phillips CM12 electron microscope at 40 kV. Length measurements were made by capturing the images with a Gatan 794 CCD camera attached to the CM12 and using Digital Micrograph 3.3 (Gatan, Pleasanton CA, USA). Images for publication were captured on sheet film and scanned with a Nikon LS4500 film scanner. The contrast was optimized, and the panels were arranged using Adobe Photoshop software.
Detection of endogenous AP sites in HeLa cells and in calf thymus DNA
HeLa S3 cells were obtained as a suspension from the Lineberger Comprehensive Cancer Center at the University of North Carolina. DNA isolation was performed using the PureGene DNA extraction kit (Gentra Systems, Minneapolis, MN, USA). Briefly, cell pellets were lysed in lysis buffer supplemented with 20 mM 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), an oxygen radical scavenger. After protein precipitation with the solution provided by the manufacturer, the DNA/RNA mixture in the supernatant was precipitated with isopropyl alcohol. The DNA/RNA pellet was resuspended in PBS buffer (pH 7.4) containing 20 mM TEMPO and incubated with RNase A (100 µg/ml) at 37°C for 30 min, followed by ethanol precipitation of the protein and DNA. The DNA pellet was resuspended in a solution that contained 1 mg/ml proteinase K, 1% SDS, and 10 mM TE and incubated for 1 h at 37°C. This method of DNA purification does not introduce any additional AP sites (4
, 5
; and data not shown). After being incubated, the samples were chromatographed through 2 ml Biogel A5M columns to remove denatured and proteolyzed proteins. Fresh calf thymus DNA was prepared as described in (3
, 9
, 10)
. AP sites in purified DNA from calf thymus and HeLa cells were identified as described above.
RESULTS AND DISCUSSION
Global detection of AP sites
To determine the endogenous amount of AP sites in genomic DNA from calf thymus or HeLa S3 cells, DNA was isolated using a method that we have shown previously not to introduce additional AP sites (5
, 11)
. To quantify AP sites, we used the ASB, which uses an ARP linked to a biotin tag (5)
to specifically react with the aldehyde that is formed when AP sites tautomerize from the closed to the open ring form. Streptavidin-conjugated horseradish peroxidase is used to generate chemiluminescence (Fig. 1
). After isolating the genomic DNA, we found the global number of AP sites determined by the ASB to be 1 site per 160–210 knt (80–105 kb) for both HeLa cell and calf thymus DNA. Thus, the approximate number of AP sites is between 60,000 and 100,000 per cell, which is in the range (2,000–200,000/cell) reported in the literature (for a review, see ref 12
).
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Detection of AP sites in control plasmids
Although the slot blot assay is a proven and accurate method to detect the global number of AP sites, it cannot detect their distribution in DNA. EM, however, in addition to detecting AP sites, allows the visualization of individual DNA fibers and accurate measurements of DNA lengths within 100–200 bp. We used EM to visualize the location of AP sites in DNA fibers derived from the same calf thymus and HeLa DNA used for the slot blot analysis. To determine the sensitivity of this technique, plasmid DNA was incubated with methoxyamine (MX) to mask any endogenous AP sites and then treated with acid and heat to produce a limited number of new AP sites (Fig. 2
A; refs 5
, 13
). Control (MX-treated) DNA and DNA samples containing newly formed AP sites were reacted with the biotinylated aldehyde reactive probe (bio-ARP) to yield a biotin moiety at each AP site. Ten-nanomolar gold particles conjugated with streptavidin were used to label each biotinylated AP site for detection by EM. Bound complexes of streptavidin/biotin were stabilized by fixation, excess gold particles were removed by gel filtration, and specimens were prepared for EM by rotary tungsten shadowcasting (Fig. 2B and C
; refs 7
, 8
). Random fields of plasmid DNA molecules were photographed, and at least 100 molecules were scored per sample. The MX-treated DNA contained the lowest number of AP sites per plasmid, whereas DNA that was exposed to heat and acid for the longest time contained the highest number of induced AP sites per plasmid (Fig. 2D
). Some plasmid molecules (3 kb) contained more than six AP sites, suggesting that our detection technique can identify AP sites within 500 bp of one another (data not shown).
|
Detection of closely spaced AP sites (AP site clusters) in isolated DNA fibers from calf thymus and HeLa cells
When calf thymus and HeLa DNA fibers were visualized, the number of AP sites detected per genomic DNA fiber varied from 0 to >30 (Fig. 3
). The AP clusters were also seen in normal human fibroblasts (NHF1; data not shown). Further analysis of 687 HeLa genomic DNA fibers (16,300,000 bp) in three independent experiments revealed that 78% of the fibers did not contain an AP site, 13% had a single AP site, 6% had between 2 and 8 AP sites, and 4% had >8 AP sites (Table 1
; Fig. 4
A). In the 4% of molecules that contained more than 8 AP sites, the number ranged from 9 to >30 with most having between 9 and 15. The average length of the fibers containing AP clusters that were measured (n=37) was 25,000 bp, and the average number of AP sites was 10.5. In these fibers, the spacing between AP sites was quite variable, ranging in distance from around 50 to thousands of bp (Fig. 4B
). However, 68% were spaced <500 bp apart. Using the number of AP sites, we measured in the total amount of genomic DNA analyzed and estimated the number of AP sites in the cell to be 110,000. This number is consistent with the slot blot analysis.
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Biological relevance of AP site clusters
Regions with multiple AP sites within a very short segment of DNA (10–20 bp) were thought to occur only as a result of ionizing radiation (14
15
16)
. From our studies, AP sites generated during normal tissue culture conditions or in calf thymus occur both as single isolated events and as closely spaced clusters, albeit not as closely spaced as in the AP clusters formed after ionizing radiation. The closely spaced clusters could be the net result of endogenous ROS produced in the cell yielding some active intermediates like those generated by exposure to gamma radiation. The occurrence of nonrandom distribution/clustering of AP sites in the genome leads us to believe that there may be regions in the genome with an increased vulnerability to ROS damage. On the basis of previous studies using UV or chemicals that preferentially produce bulky adducts (such as benzo(a)-pyrene diol epoxide; ref 17
) or UV, we would predict that these vulnerable regions are in areas undergoing replication, transcription, and/or between nucleosomes (18
19
20)
. An uneven distribution of AP sites also may imply that the detrimental effects of ROS in the development of disease may be related not simply to the total number of AP sites present but to how AP sites are distributed along a DNA fiber and, perhaps, to the genomic sites affected.
ACKNOWLEDGMENTS
We thank Bruna Brylawski for the critical reading of this manuscript. This work was supported by National Institutes of Health Grant CA-084493 to D. Kaufman. P. Chastain is supported by Training Grant ES07017 from the National Institute of Environmental Health Sciences. J. Nakamura and J. S Swenberg are supported by CEHS Center Grant P30-ES10126 and SFBR Grant P42-ES05948. EM studies were done in the Lineberger Cancer Center EM core facility supported by CA16086 and a grant to Jack Griffith (GM31819).
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication April 20, 2006. Accepted for publication July 24, 2006.
REFERENCES
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