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Measurement of Oxidative DNA Damagein the Human p53 and PGK1 Gene atNucleotide Resolutiona

HENRY RODRIGUEZb,c AND STEVEN A. AKMANd

bBiotechnology Division, National Institute of Standards and Technology,Gaithersburg, Maryland 20899, USAdDepartment of Cancer Biology, Comprehensive Cancer Center of Wake Forest University, Winston-Salem, North Carolina 27157, USA

INTRODUCTION

DNA damage induced by ROSe is an important intermediate in the pathogenesisof human conditions such as cancer and aging.1 The mutational spectra of H2O2

2 andthe transition metal ions Fe, Cu,3,4 and Cr have been studied in model systems, butthe relationship of induced DNA damage to these spectra remains unknown.

We mapped ROS-induced DNA base modifications sensitive to Nth and Fpg pro-teins.6 Using a modified ligation-mediated polymerase chain reaction (LMPCR)technique, we investigated the in vivo and in vitro frequencies of DNA base modifi-cations caused by ROS in the human p53 and PGK1 genes.6–8

MATERIALS AND METHODS

In Vivo H2O2 Treatment of Human Skin Fibroblasts. Human male foreskin fibro-blasts were treated, harvested, and the DNA isolated as previously described.9

In Vitro Metal Ion-Ascorbate-H2O2 Treatment. Dialyzed DNA was treated withCuCl2, FeCl3, or K2Cr2O7 as previously described.8

Ligation-Mediated Polymerase Chain Reaction. Digestion of treated DNA withNth and Fpg proteins and the LMPCR technique (FIG. 1) has been described in detailelsewhere.6 Key steps are:

aCertain commercial equipment, instruments, materials, or companies are identified in thispaper to specify adequately the experimental procedure. Such identification does not imply rec-ommendation or endorsement by the National Institute of Standards and Technology, nor does itimply that the materials or equipment identified are the best available for the purpose.

cAddress for correspondence: Dr. Henry Rodriguez, Biotechnology Division, National Insti-tute of Standards and Technology, 100 Bureau Drive, Stop 8311, Gaithersburg, Maryland 20899-8311. Phone: 301-975-2578; fax: 301-975-8505.

e-mail: henry.rodriguez@nist.govWeb: http://www.nist.goveABBREVIATIONS: PGK1, PhosphoGlycerate Kinase; LMPCR, ligation-mediated polymerase

chain reaction; ROS, reactive oxygen species; H2O2, hydrogen peroxide; Fpg, FormamidoPyrim-idine DNA glycosylase; Nth, Endonuclease III.

383 RODRIGUEZ & AKMAN: OXIDATIVE DNA DAMAGE

(1) conversion of a modified base into a strand break, chemically or enzymati-cally, followed by primer extension of an upstream primer 1 to generateblunt ends;

(2) ligation of a universal asymmetric double-strand linker;(3) PCR amplification using a second upstream primer 2 with a downstream

linker primer; and(4) separation of DNA fragments on a sequencing gel, transfer to a nylon

membrane, and hybridization with a radiolabeled probe.

CONCLUSION

The distribution of oxidative damage induced in exons 5 and 9 of human p53 andthe promoter region of human PGK1 gene was assessed. A representative autoradio-

FIGURE 1. Schematic representation of the steps in DNA base damage mapping byLMPCR.

384 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. LMPCR analysis of damage induced in the promoter region of humanPGK1 using primer set A (transcribed strand). Lanes 1–4, DNA treated with standard Max-am-Gilbert cleavage reactions. Lanes 5–6, 13, DNA recovered from intact human foreskinfibroblasts exposed to 50 mM H2O2. Lanes 7–8, 14, dialyzed genomic DNA treated with 100µM Fe(III)/100 µM ascorbate/5 mM H2O2 in the presence of 0.3 M sucrose. Lanes 9–10,15, DNA treated with 50 µM Cu(II)/100 µM ascorbate/5 mM H2O2 in the presence of 1 mMpotassium phosphate buffer. Lanes 11–12, 16, DNA treated with 100 µM Cr(VI)/100 µMascorbate/5 mM H2O2. Lane 17, DNA incubated in potassium phosphate buffer and digestedwith Nth and Fpg proteins. The DNA in lanes 5–12 was digested with Nth and Fpg proteins

385 RODRIGUEZ & AKMAN: OXIDATIVE DNA DAMAGE

gram indicating the damage distributions induced in the PGK1 gene is shown inFIGURE 2.

The nucleotide-resolution maps of DNA base damage induced in vitro in the pres-ence of Cu(II), Fe(III), or Cr(VI) transition metal ions were similar to the in vivobase damage induced by H2O2. The in vitro similarity suggest a model in which thelocal binding site occupancy rate and the local geometry of the metal ion-DNA-per-oxo coordination complex determine the damage event. The principal determinantof a damaging event occurring at any position is DNA sequence context. Guaninewas the most heavily modified base. The triplet d(pCGC) was the principal hotspotsequence with guanine stretches also hit.8

REFERENCES

1. AMES, B.N. 1987. Oxidative DNA damage, cancer, and aging. Ann. Intern. Med. 107:526–545.

2. MORAES, E.C., S.M. KEYSE, M. PIDOUX & R.M. TYRRELL. 1989. The spectrum ofmutations generated by passage of a hydrogen peroxide damaged shuttle vector plas-mid through a mammalian host. Nucleic Acids Res. 17: 8301–8312.

3. AKMAN, S.A., G.P. FORREST, J.H. DOROSHOW & M. DIZDAROGLU. 1991. Mutation ofpotassium permanganate and hydrogen peroxide-treated plasmid pz189 replicating incv-1 monkey kidney cells. Mutat. Res. 261: 123–130.

4. LOEB, L.A., E.A. JAMES, A.M. WALTERSDORPH & S.J. KLEBANOFF. 1988. Mutagenesisby the autoxidation of iron with isolated DNA. Proc. Natl. Acad. Sci. USA 85: 3918–3922.

5. TKESHELASNVILI, L.K., T. MCBRIDE, K. SPENCE & L.A. LOEB. 1991. Mutation spec-trum of copper-induced DNA damage. J. Biol. Chem. 266: 6401–6406.

6. RODRIGUEZ, H., R. DROUIN, G.P. HOLMQUIST, T.R. O’CONNOR, S. BOITEUX, J. LAVAL,J.H. DOROSHOW & S.A. AKMAN. 1995. Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J. Biol. Chem. 270: 17633–17640.

7. RODRIGUEZ, H., R. DROUIN, G.P. HOLMQUIST & S.A. AKMAN. 1997. A hot spot forhydrogen peroxide-induced damage in the human hypoxia-inducible factor 1 bindingsite of the PGK 1 gene. Arch. Biochem. Biophys. 338: 207–212.

8. RODRIGUEZ, H., G.P. HOLMQUIST, R. D’AGOSTINO, Jr., J. KELLER & S.A. AKMAN. 1997.Metal ion-dependent hydrogen peroxide-induced DNA damage is more sequencespecific than metal specific. Cancer Res. 57: 2394–2403.

9. DROUIN, R., H. RODRIGUEZ, S.W. GAO, Z. GEBREYES, T.R. O’CONNOR, G.P. HOLM-QUIST & S.A. AKMAN. 1996. Cupric ion ascorbate hydrogen peroxide-induced DNAdamage: DNA-bound copper ion primarily induces base modifications. Free RadicalBiol. Med. 21: 261–273.

after treatment; the DNA in lanes 13–16 was incubated in digestion buffer alone after treat-ment. Positions of high damage frequency bases are marked with arrows to the left of lane1. The sequence of positions heavily damaged in the presence of chromium, but not copperor iron, is denoted by rectangles to the right of lane 12.