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Endogenous Apurinic/Apyrimidinic Sites in Genomic DNA of Mammalian Tissues¹

Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, North Carolina 27599

	ABSTRACT

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Apurinic/apyrimidinic (AP) sites are one of the most frequent lesions in DNA. Using a highly sensitive slot blot assay, we determined the number and condition of endogenous AP sites in normal tissues of rats and human liver. The number of AP sites (50,000–200,000 per mammalian cell) was greatest in brain, followed by colon and heart, and then liver, lung, and kidney. The majority of endogenous AP sites were cleaved 5' to the AP site. These data suggest that removal of the deoxyribosyl phosphate moiety is the rate-limiting step in base excision and AP site repair in vivo.

	Introduction

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Cellular DNA is continuously exposed to insults from exposure to endogenous and exogenous alkylating agents, ionizing radiation, and oxidative stress. It has been proposed that DNA base damage generated by these agents is repaired mainly by base excision repair pathways (1) . This repair process is initiated by spontaneous depurination or DNA glycosylase-mediated hydrolytic cleavage at the N-glycosyl bond between the base and deoxyribose, resulting in the formation of an AP³ site. The spontaneous depurination rate was estimated at 10,000 bases per cell per day under physiological conditions by extrapolation of the data obtained upon release of radiolabeled bases from DNA at 70°C (2) . Recently, using a combination of an ARP and slot blot techniques (ASB assay), we directly demonstrated the spontaneous depurination rate under physiological conditions to be 1.5 AP sites per 10⁶ nucleotides per day, which corresponds to 9000 AP sites per cell per day (3) . These experiments suggest that spontaneous depurination is one of the most frequent promutagenic events in genomic DNA. Furthermore, direct release of bases is also induced by free radical attack of the deoxyribose (4) . These AP sites have to be repaired efficiently because of their potential cytotoxicity and mutagenicity (5) . To date, the kinetics of AP site repair have been studied in detail only in in vitro experiments with oligonucleotides and plasmids. The steady-state number of AP sites in genomic DNA has not been characterized because of technical problems related to sensitivity and specificity of existing methods. To better understand the steady state of AP sites at basal levels of base excision repair in vivo, we measured the amount of endogenous AP sites in genomic DNA extracted from various tissues of adult rats and human liver using the ASB assay. In addition, the endogenous AP sites were further characterized by the determination of cleavage sites to the AP sites in these genomic DNAs.

	Materials and Methods

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DNA Extraction
Six male F344 rats were obtained from Charles River Laboratories and maintained until they were 7.5 months old. Rats were sacrificed after being anesthetized with metofane. Tissues and organs were quickly harvested and frozen at -80°C until DNA extraction. Eight human liver samples were provided by Dr. C. J. Omiecinski (University of Washington, Seattle, WA; Ref. 6 ). DNA was extracted by a procedure slightly modified from the method reported by Nakamura et al. (3) . Briefly, frozen tissues were thawed and homogenized in PBS with a Tehran homogenizer (Wheaton Instruments, Millville, NJ). After centrifugation at 2,000 x g for 10 min, the nuclear pellets were incubated in lysis buffer (Applied Biosystems) overnight at 4°C with proteinase K (500 mg/ml; Applied Biosystems). DNA was then extracted twice with a mixture of phenol, chloroform, and water and once with Sevag, followed by ethanol precipitation. The extracted DNA was incubated in PBS (pH 7.4) with a mixture of RNase and RNase A. After DNA precipitation with cold ethanol, the DNA pellet was resuspended in sterilized distilled water. The DNA solution was stored at -80°C for the ASB assay. The DNA extraction method used in this study was unlikely to have modified the original number of AP sites in genomic DNA from intact tissues, based on reextraction data of DNA highly exposed to methylmethane sulfonate.

ASB Assay
The AP site assay was performed by a procedure slightly modified from the method reported by Nakamura et al. (3) . Briefly, 15 µg of DNA in 150 µl of PBS were incubated with 1 mM ARP (Dojindo Laboratories, Kumamoto, Japan) at 37°C for 10 min. After precipitation using cold ethanol, DNA was resuspended in Tris-EDTA buffer. The DNA concentration was measured by a spectrophotometer, and the DNA solution was then prepared at 1.5 µg per 100 µl of Tris-EDTA buffer. Heat-denatured DNA was then immobilized on a NC membrane. The NC membrane was soaked with 5x SSC and then baked in a vacuum oven. The membrane was preincubated with 10 ml of Tris-HCl buffer containing BSA. The NC filter was then incubated in the same solution containing streptavidin-conjugated horseradish peroxidase (BioGenix) at room temperature for 40 min. After the NC membrane was rinsed, the enzymatic activity on the membrane was visualized by the use of enhanced chemiluminescence reagents (Amersham Corp.). The NC filter was then exposed to X-ray film, and the developed film was analyzed using an Ultrascan XL scanning densitometer.

Heat/Acid Treatment of Calf Thymus DNA
Calf thymus DNA was treated with 100 mM MX in 10 mM Tris-HCl buffer-KOH (pH 7.4) at 37°C for 2 h to reduce the original number of AP sites in calf thymus DNA (Sigma). The DNA was purified twice by ethanol precipitation, followed by resuspension in distilled water. The calf thymus DNA pretreated with MX was incubated with heat/acid buffer as described in a previous paper (3) for various periods of time.

AP Site Cleavage Assay
Regular AP Site Assay.
Fifteen µg of DNA in 135 µl of 10 mM Tris-HCl-KOH buffer (pH 7.5) containing 50 mM NaCl and 5 mM MgCl₂ were incubated with 1 mM ARP at 37°C for 10 min, followed by the ASB assay described above.

5' Cleavage Assay.
Fifteen µg of DNA and 145 units of Escherichia coli Exo III (New England Biolabs) in 135 µl of 10 mM Tris-HCl-KOH buffer as described above were incubated at 37°C for 1 min, immediately followed by the ASB assay. The number of 3'-cleaved AP sites was calculated as the original number of AP sites minus the number of AP sites left after treatment with Exo III alone.

3' Cleavage Assay.
Fifteen µg of DNA, 10 mM EDTA, and 100 mM putrescine (Sigma) in 135 µl of 10 mM Tris-HCl-KOH buffer as described above were incubated at 37°C for 30 min, immediately followed by the ASB assay. The number of 5'-cleaved AP sites was calculated as the original number of AP sites minus the number of AP sites left after treatment with putrescine alone.

Detection of Residual AP Sites.
Fifteen µg of DNA and 145 units of Exo III in 110 µ1 of 10 mM Tris-HCl-KOH buffer as described above were incubated at 37°C for 1 min, immediately followed by the addition of a 1/10 volume of 100 mM EDTA. The sample was incubated with 100 mM putrescine in the reaction buffer at 37°C for 30 min, immediately followed by the ASB assay. Residual AP sites showed the number of uncleaved aldehydic lesions after the combination treatment of Exo III and putrescine.

Depurination Assay
Fifteen µg of DNA in 110 µl of PBS (pH 7.4) were incubated at 100°C for 20 min, followed by renaturation at room temperature for 1 h. After incubation with 100 mM putrescine, DNA was reacted with ARP, followed by the ASB assay. The number of heat-labile sites was calculated as the number of AP sites in DNA treated with heat buffer followed by putrescine minus the number of AP sites left after treatment with putrescine alone.

End III-sensitive Assay
Oxidative pyrimidine bases are repaired by End III, leaving AP sites on DNA backbone (5) . E. coli End III was kindly provided by Dr. Y. W. Kow (Emory University, Atlanta, GA). Fifteen µg of DNA, 1 mM EDTA, 100 mM NaCl, 100 mM putrescine, and E. coli End III (0.1 µg) in 135 µl of 10 mM Tris-HCl-KOH buffer as described above were incubated at 37°C for 30 min, immediately followed by the ASB assay. The number of End III-sensitive sites was calculated as number of AP sites in DNA treated with End III and putrescine minus the number of AP sites left after treatment with putrescine alone.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
To better understand the formation and repair of AP sites resulting from base excision repair and spontaneous depurination/depyrimidination in vivo, we measured the number of endogenous AP sites in genomic DNA extracted from various tissues of adult rats and transplant-quality human liver using the ASB assay (3) . The number of endogenous AP sites varied widely between tissues (Fig. 1) but not within tissues. The highest number of AP sites was detected in the brain, with 30 AP sites per 10⁶ nucleotides, followed by the heart and colon. In contrast, rat liver, kidney, and lung consistently showed the lowest number of AP sites (8–9 AP sites per 10⁶ nucleotides). The number of endogenous AP sites in human liver was comparable to that in rat liver. These data indicate that AP sites persist at 50,000–200,000 lesions per mammalian cell under normal physiological conditions. The steady-state number of AP sites in genomic DNA should reflect the balance between the formation and repair of the AP sites. Possible interpretations of the higher number of endogenous AP sites in the brain are as follows: (a) higher rate of depurination and/or endogenous DNA adduct formation, resulting in more AP sites; (b) higher activity of DNA glycosylase, inducing more AP sites; (c) lower activity of type II AP endonuclease, causing accumulation of AP sites; and (d) less efficiency of dRp-ase or ß-elimination, leaving 5'-nicked AP sites.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Endogenous AP sites in rat and human tissues. DNA was extracted at 4°C from intact rat tissues and normal human livers, and the number of AP sites was measured using the ASB assay. A, typical X-ray film showing endogenous AP sites of rat tissues. DNA (including standard DNA samples) was loaded on a NC membrane (1.5 µg per slot). B, scanning densitometric data of endogenous AP sites in rat and human tissues. Columns, means from duplicate slots of five to eight individual samples; bars, SD.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Scheme of AP site cleavage assay. To better understand the activities of type II AP endonuclease, as well as dRp-ase in vivo, we determined the existence of cleavages on the 5' or 3' side of AP sites in genomic DNA. Putrescine and Exo III (type II AP endonuclease) leave 3'-cleaved [(II)] and 5'-cleaved [(III)] AP sites, respectively. For intact AP sites [(I)] with no incision at the 3' and 5' sides of AP sites, the ASB assay can theoretically detect the original number of AP sites after treatment with either Exo III [(III)] or putrescine [(II)] because AP sites remain on the DNA backbone after incision at either side of the phosphodiester bond adjacent to the AP site. However, the combination of Exo III and putrescine cleaves at both 3' and 5' sides of the AP site [(IV)], resulting in release of the AP site from the DNA backbone [(V)]. Such released AP sites are not detected by this assay. On the basis of the number of AP sites left on the DNA backbone after this cleavage reaction, the number of 5'- and 3'-cleaved AP sites can be estimated. If 5'-cleaved AP sites [(VI)] are present in DNA, the putrescine single treatment can release the 5'-nicked AP sites by 3'-excision of the AP sites [(VII) and (VIII)]. Likewise, 3'-nicked AP sites [(IX)] in DNA can be released by 5'-excision using Exo III [(X) and (XI)].

View larger version (28K): [in this window] [in a new window]   Fig. 3. AP site cleavage assay of calf thymus DNA treated with heat/acid buffer. To validate the AP site cleavage assay, we examined the effects of Exo III and putrescine on intact AP sites induced in calf thymus DNA. The original number of AP sites in calf thymus DNA was reduced by MX (CTD/MX). DNA was then incubated in heat/acid buffer for different lengths of time to introduce different numbers of intact AP sites (-/-; Ref. 9 ). DNA was incubated with Exo III and/or putrescine, and the number of remaining AP sites in calf thymus DNA was measured by the ASB assay [Exo III only (Exo III/-), Exo III plus putrescine (Exo III/P), and putrescine only (-/P)]. Columns, means from duplicate slots of three individual samples; bars, SD. A, AP site cleavage assay of DNA containing 115 AP sites per 106 nucleotides. B, summary of AP site cleavage assay of DNA containing different numbers of AP sites.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Summary of AP site cleavage assay of rat and human tissue DNA. The original number of AP sites as described in the legend to Fig. 1B consisted of 5'-cleaved, 3'-cleaved, intact AP sites, and residual aldehydic lesions. The number of 5'-cleaved AP sites was calculated as the original number of AP sites minus the number of AP sites left after treatment with putrescine alone. The combined fraction of 3'-cleaved and intact AP sites was the difference between putrescine treatment and the combination treatment of Exo III and putrescine. Residual AP sites showed the number of uncleaved aldehydic lesions.

In the previous study (3) , we demonstrated that spontaneous depurination occurred at 1.5 AP sites per 10⁶ nucleotides per day under physiological conditions. To test whether heat-labile DNA adducts such as N3- and N7-alkyl purines were the source of the higher number of endogenous AP sites in brain, a depurination assay was performed in brain and liver DNA. However, no difference was observed in brain and liver (data not shown). Therefore, the steady state of endogenous AP sites may not be due to labile base lesions. One of the most important and abundant endogenous DNA lesions is oxidative DNA base damage. It has been reported that the steady state of 5-hydroxycytosine in rat brain is 2-fold higher than that in rat liver (19) . Such oxidative pyrimidine bases are repaired by End III, leaving AP sites on the DNA backbone. To examine whether oxidative stress could be related to the steady-state number of AP sites in DNA, End III-sensitive sites were quantitated using the combination of the ASB assay and E. coli End III. The number of End III-sensitive sites was three times higher in brain (14 lesions per 10⁶ nucleotides) compared to other tissues (4–5 lesions per 10⁶ nucleotides). Recently, the human homologue of the End III (hNTH1) gene has been cloned and characterized (20) . The mRNA expression of this gene varies between organs in a pattern that corresponds with the number of endogenous AP sites (20) . Most DNA glycosylases involved in the repair of oxidized bases possess AP lyase activity, which cleaves the 3' side of AP sites. Therefore, endogenous 5' AP sites would not be derived from the cleavages of oxidative bases by such DNA glycosylases. A preliminary experiment showed that hydrogen peroxide with FeSO₄ directly induced 5'-cleaved AP sites in calf thymus DNA without any enzymes (data not shown). These studies suggest that oxidative stress might be one of the factors responsible for the steady state of AP sites with 5' cleavage.

We originally expected to find low numbers of endogenous AP sites in genomic DNA because of their toxicity and mutagenicity. However, this study shows that the steady state of AP sites is 1 lesion per 10⁵ nucleotides in genomic DNA. Although AP sites are constantly being repaired, the fraction of AP sites that escapes repair is likely to contribute to mutations, chromosome aberrations, and transcription errors that may be associated with spontaneous age-related diseases such as cancer and degenerative disorders.

	ACKNOWLEDGMENTS

 
We thank C. J. Omiecinski, C. Hassett, and K. Thummel for providing the human liver samples; Y. W. Kow for providing End III; and A. Lindstorm for providing F344 rats. We also thank E. C. Kidd, P. B. Upton, K. S. McDorman, and N. Scheller for assisting in the preparation of the manuscript and for technical assistance. The critical reading of the manuscript by M. D. Topal, S. A. Leadon, W. K. Kaufmann, A-J. L. Ham, A. P-H. Lin, and D. K. La is acknowledged.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Funded in part by National Institute of Environmental Health Sciences Superfund Basic Research Program Grant P42-ES05948.

² To whom requests for reprints should be addressed, at Department of Environmental Sciences and Engineering, The University of North Carolina, CB 7400, Chapel Hill, NC 27599-7400.

³ The abbreviations used are: AP, apurinic/apyrimidinic; ARP, aldehyde-reactive probe; ASB, ARP-slot blot; NC, nitrocellulose; MX, methoxyamine; Exo III, exonuclease III; End III, endonuclease III; 5'-dRp, deoxyribose-5-phosphate; dRp-ase, 5'-deoxyribophosphodiesterase; ß-pol, polymerase ß.

Received 2/ 1/99. Accepted 4/15/99.

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				M. Guillet and S. Boiteux Origin of Endogenous DNA Abasic Sites in Saccharomyces cerevisiae Mol. Cell. Biol., November 15, 2003; 23(22): 8386 - 8394. [Abstract] [Full Text] [PDF]

				A. Barbin, H. Ohgaki, J. Nakamura, M. Kurrer, P. Kleihues, and J. A. Swenberg Endogenous Deoxyribonucleic Acid (DNA) Damage in Human Tissues: A Comparison of Ethenobases with Aldehydic DNA Lesions Cancer Epidemiol. Biomarkers Prev., November 1, 2003; 12(11): 1241 - 1247. [Abstract] [Full Text]

				K. L. Meadows, B. Song, and P. W. Doetsch Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: implications for biological function Nucleic Acids Res., October 1, 2003; 31(19): 5560 - 5567. [Abstract] [Full Text] [PDF]

				D. Wong, M. S. DeMott, and B. Demple Modulation of the 3'->5'-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5'-incised Abasic DNA Product J. Biol. Chem., September 19, 2003; 278(38): 36242 - 36249. [Abstract] [Full Text] [PDF]

				L. Calderon-Garciduenas, R. R. Maronpot, R. Torres-Jardon, C. Henriquez-Roldan, R. Schoonhoven, H. Acuna-Ayala, A. Villarreal-Calderon, J. Nakamura, R. Fernando, W. Reed, et al. DNA Damage in Nasal and Brain Tissues of Canines Exposed to Air Pollutants Is Associated with Evidence of Chronic Brain Inflammation and Neurodegeneration Toxicol Pathol, August 1, 2003; 31(5): 524 - 538. [Abstract] [PDF]

				P.-H. Lin, J. Nakamura, S. Yamaguchi, S. Asakura, and J. A. Swenberg Aldehydic DNA lesions induced by catechol estrogens in calf thymus DNA Carcinogenesis, June 1, 2003; 24(6): 1133 - 1141. [Abstract] [Full Text] [PDF]

				H. Kamiya Mutagenic potentials of damaged nucleic acids produced by reactive oxygen/nitrogen species: approaches using synthetic oligonucleotides and nucleotides: SURVEY AND SUMMARY Nucleic Acids Res., January 15, 2003; 31(2): 517 - 531. [Abstract] [Full Text] [PDF]

				J. R. Silber, M. S. Bobola, A. Blank, K. D. Schoeler, P. D. Haroldson, M. B. Huynh, and D. D. Kolstoe The Apurinic/Apyrimidinic Endonuclease Activity of Ape1/Ref-1 Contributes to Human Glioma Cell Resistance to Alkylating Agents and Is Elevated by Oxidative Stress Clin. Cancer Res., September 1, 2002; 8(9): 3008 - 3018. [Abstract] [Full Text] [PDF]

				A. G. Georgakilas, P. V. Bennett, and B. M. Sutherland High efficiency detection of bi-stranded abasic clusters in {gamma}-irradiated DNA by putrescine Nucleic Acids Res., July 1, 2002; 30(13): 2800 - 2808. [Abstract] [Full Text] [PDF]