ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 338, No. 2, February 15, pp. 207–212, 1997Article No. BB969820

A Hot Spot for Hydrogen Peroxide-Induced Damagein the Human Hypoxia-Inducible Factor 1 BindingSite of the PGK 1 Gene1

Henry Rodriguez,*,2 Regen Drouin,†,2,3 Gerald P. Holmquist,† and Steven A. Akman*,4

*Department of Medical Oncology and †Division of Biology, Beckman Research Institute,City of Hope National Medical Center, Duarte, California 91010

Received June 4, 1996, and in revised form November 25, 1996

day in humans (1) and increasing in extent and rate ofUsing ligation-mediated polymerase chain reaction formation with age (2). A principal source of ROS is

to separately map the distribution of induced oxidized H2O2, a weak mutagen (3) and a metabolic by-productbases and strand breaks along the human PGK1 pro- of oxidative phosphorylation. It is produced in mito-moter at nucleotide resolution, we previously de- chondria at the rate of about 1 mol of H2O2 per 50 molscribed the pattern of oxidative DNA damage induced of O2 consumed and, if not degraded, may diffuse toin vitro by Cu(II)/ascorbate/H2O2 [J. Biol. Chem. 270, the nucleus where it oxidatively stresses the genome17633–17640 (1995)]. Here we report that the pattern (4). ROS-induced DNA damage has been implicated inof in vivo base damage caused by H2O2 is almost identi- causing endogenous mutations (5) which lead to cancercal to that of the previously used in vitro system with (6). However, there are as yet no data addressing thethe exception of transcription factor-associated foot- distribution of ROS-induced DNA damage or the repairprints. An unusually strong positive footprint for both of such damage at nucleotide resolution in target geno-strand breaks and oxidized bases is associated with mic genes. Such data will be crucial to establishing abinding of the hypoxia-inducible transcription factor-

link between ROS-induced DNA damage and muta-1. Base damage at this footprint was 52–91% repairedtional spectra derived from tumor-associated genes inin 24 h, which was similar to the global base damagehuman neoplasms.repair rate. However, strand breaks at this footprint

Ligation-mediated PCR (LMPCR) is an extremelywere only 39–55% repaired in 24 h or approximatelysensitive method for mapping the frequency of rare100-fold slower than the global strand break repairDNA breaks along a complex genome. A primer is ex-rate. q 1997 Academic Press

tended to the breaks, creating blunt double-strandedKey Words: DNA base damage; DNA strand breaks;ends. After blunt end ligation of an assymetric double-DNA repair; reactive oxygen species; footprints.stranded linker, a population of molecules whose sizereflects the distance from the primer pairing site to thebreaks is amplified by PCR, separated by sequencing

Oxidative DNA damage is induced by ROS,5 oc- gel electrophoresis, transferred to a nylon membrane,curring at the rate of about 10,000 events per cell per and probed, and the resulting autoradiogram is ana-

lyzed. By treating permeabilized cells with DNase I toinduce breaks or by irradiating the cells with uv light1 This work was supported by Public Health Service Grant

CA53115 from the National Cancer Institute. and subsequently cleaving the DNA at the photole-2 These authors contributed equally to this work. sions, DNase I footprints (7) and photo-footprints (8)3 Present address: Unite de Recherche en Genetique Humaine et due to transcription factor binding were mapped by

Moleculaire, Centre de Recherche, Hopital St.-Francois d’Assise,LMPCR along the human PGK1 promoter. PGK1 is anQuebec (Quebec), Canada G1L 3L5.X-linked gene and the footprints were absent from the4 To whom correspondence should be addressed at Department of

Cancer Biology, Wake Forest Comprehensive Cancer Center, Medi- allele on the inactive X chromosome (7, 8).cal Center Blvd., Winston–Salem, NC 27157. Fax: (910) 716-0255. We have recently adapted the LMPCR technique toE-mail: sakman@bgsm.edu. map the frequency of oxidative DNA damage at nucleo-5 Abbreviations: ROS, reactive oxygen species; LMPCR, ligation-

tide resolution in target genes (9). Ligatable DNAmediated polymerase chain reaction; HIF-1, hypoxia inducible factor1; DMEM, Dulbecco’s modified essential medium. strand breaks corresponding to certain oxidatively

2070003-9861/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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as follows: (i) primer extension of an annealed gene-specific oligonu-modified pyrimidines are created by digesting damagedcleotide (upstream primer 1) to generate blunt ends; (ii) ligation ofDNA with Nth protein, a DNA–N-glycosylase/(apuri-a universal asymmetric double-strand linker onto the blunt ends;nic/apyrimidinic) endonuclease purified from Esche- (iii) PCR amplification using a second gene-specific oligonucleotide

richia coli; breaks corresponding to oxidatively modi- (upstream primer 2) along with a linker primer (downstream linkerprimer); (iv) separation of the DNA fragments on a sequencing poly-fied purines are created by digestion with the E. coliacrylamide gel; (v) transfer of the DNA to a nylon membrane byFpg protein [see Ref. (10) for a review of these en-electroblotting; and (vi) hybridization of a radiolabeled probe pre-zymes]. The majority of direct DNA strand breakspared by repeated primer extension using a third gene-specific oligo-

caused by reactive oxygen species are ligatable and nucleotide (upstream primer 3).therefore are detectable by LMPCR without further Autoradiograms. Air-dried membranes were exposed to Kodakmodification (9). In this report, we determine the fre- XAR-5 X-ray films for 0.5 to 8 h with intensifying screens at 0707C.

On the final autoradiogram, each band represents a nucleotide posi-quency pattern of H2O2-induced oxidative DNA damagetion where a break was induced, and the signal intensity of the bandin a portion of the PGK1 gene of human fibroblasts.reflects the number of DNA molecules with ligatable ends terminat-Footprints of H2O2-induced damage are mapped by ing at that position. The intensity of the bands was confirmed by

comparison with the damage frequency patterns in- phosphorimager (Molecular Dynamics, Inc.) scans.duced in purified human genomic DNA in vitro by thetransition metal ion-catalyzed Fenton reaction.

RESULTS

In the presence of ascorbate, Cu(II) in solution isMATERIALS AND METHODSreduced to Cu(I). Cu(I) binds tightly to DNA and formsIn vitro treatment of human genomic DNA with Cu(II)/ascorbate/a DNA–Cu(I)–H2O2 complex which, when resolved byH2O2. Human male fibroblast genomic DNA was isolated and dia-

lyzed extensively against distilled water as previously described (11). H2O2-mediated oxidation of the Cu(I) to Cu(II), causesTen-microgram aliquots of dialyzed fibroblast DNA were exposed to DNA base damage with little strand cleavage (11). The50 mM CuCl2, 100 mM ascorbate, 5 mM H2O2, in 1 mM potassium base damage includes ring-saturated, ring-contracted,phosphate buffer, pH 7.5, for 30 min at 377C. After exposure, EDTA

and ring-fragmented pyrimidines as well as imidazolewas added to 1 mM, and the DNA was ethanol precipitated, washed,and dissolved in Nth/Fpg digestion buffer. ring-open and C8-oxo purines (13). The E. coli base

In vivo treatment with H2O2 and/or ultraviolet B light. Confluent excision repair proteins Fpg (formamidopyrimidineprimary human foreskin fibroblasts in Dulbecco’s modified essential glycosylase) and Nth (endonuclease III) together cleavemedium (DMEM) plus 10% fetal bovine serum ({additional 50 mM DNA at these oxidized bases producing 5* Pi ends (14–CuCl2 for 16 h immediately prior to H2O2 exposure) were treated in

16), the substrates for LMPCR.vivo with 50 mM H2O2 for 30 min at 377C in serum-free DMEM toWe evaluated the frequency pattern of DNA damageproduce, without added CuCl2, 0.076 breaks/kb and 0.33 modified

bases/kb. After H2O2 exposure, cell monolayers were rinsed in phos- in the promoter region of human PGK1, a gene on thephate-buffered saline, then DNA was isolated as described (9) for X chromosome which codes for the enzyme phospho-LMPCR analysis. glycerol kinase. This region of the genome was chosenTo determine whether chromatin structure was perturbed follow-

because the position of bound transcription factors hasing the 30-min H2O2 exposure and rinsing with cold 154 mM NaCl,been well characterized (7, 8). The frequency patternfibroblasts were irradiated with 10,000 J/m2 of ultraviolet B light.

This dose of ultraviolet B light generates an average of one cyclopyri- of base damage induced by Cu(II)/ascorbate/H2O2 in themidine dimer per kilobase of genomic DNA in intact human fibro- PGK1 promoter is almost identical to the base damageblasts. Both purified genomic DNA (65 mg/ml) and intact cells were frequency pattern induced by the treatment of cells inirradiated on ice suspended in 10 mM Tris–HCl, pH 7.4, 150 mM KCl,

vivo with H2O2 (Fig. 1). Supplementation of cellular10 mM NaCl, 1 mM EDTA. The ultraviolet B light source consisted ofcopper ion content by addition of high-concentrationtwo tubes (FS20T12/UVB/BP, Philips) delivering 7.45 J/s filtered

through a cellulose acetate screen (Kodacel TA-407 clear 0.015 in., Cu(II) to the growth medium did not alter the in vivoEastman–Kodak) to block transmission below 290 nm. base damage frequency pattern (Fig. 1); damage fre-

Enzyme digestions. Ten-microgram aliquots of DNA were di- quency was enhanced proportionally at each base posi-gested in 400 ng Fpg protein (formamidopyrimidine glycosylase) from

tion by copper ion supplementation. This similarity,E. coli and 100 ng Nth protein (endonuclease III) from E. coli, in awith õ10% of bases having nonidentical damage sig-volume of 100 ml at 377C for 60 min. These conditions produceú90%

cleavage at each position of damage products recognized by these nals in vivo and in vitro, may be true for almost allenzymes (9). After termination of the digestions, the DNA pellets regions not protected from cleavage by DNase I by tran-were dissolved with either the mix for glyoxal gel or Sequenase buffer scription factors. It has been observed in all regions of(40 mM Tris–HCl, pH 7.7, and 50 mM NaCl) as previously described

the human foreskin fibroblast genome we have exam-(9, 11).ined to date, covering 800 bases, including exons 5 andCyclopyrimidine dimers were cleaved and converted to single-

strand breaks with T4 endonuclease V (8). The ligation-inhibiting 5* 9 (transcribed strand) of the p53 gene and bases 0370pyrimidine overhang was removed by photoreactivation using E. coli to /140 (both strands) of the PGK1 promoter and firstphotolyase (8). exon (29). While the in vivo base damage pattern could

Sequencing standards. Human DNA was chemically cleaved for be due to redox cycling of endogenous DNA-bound cop-A, A / G, T, and T / C standards with formic acid, hydrazine, andper ions, which are reported to be present in chromatinpiperidine as described by Pfeifer and Riggs (12).at about one copper ion per kilobase (17), the patternLMPCR. This technique has been described elsewhere (9).

Briefly, the LMPCR procedure is a six-step process. The steps are also mimics the base damage pattern produced in vitro

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HOT SPOT FOR HYDROGEN PEROXIDE-INDUCED DNA DAMAGE 209

C0213 , C0212 , and C0202 , Fig. 2a), representing mainlypositive footprints. These are coincident with transcrip-tion factor binding sites as previously determined byother footprinting techniques (7, 8). The iron–EDTAcatalyzed Fenton reaction has historically been used tovisualize in vitro footprints (18). Free hydroxyl radical

FIG. 1. In vitro and in vivo oxidative DNA damage patterns alongthe nontranscribed strand of the human PGK1 promoter positions0123 to 057 (5*-C0123CGC GATGGGCTGT GGCCAATAGC GGC-TGCTCAG CAGGGCGCGC CGAGAGCAGC GGCCGGGAAGGGG057) using primer set F (see Ref. 9 for primer set construction).As assessed by neutral denaturing agarose gel electrophoresis (27),the untreated DNA had 0.02 strand breaks/kb, 0.003 abasic sites/kb, and no detectable modified bases (data not shown). After in vitroCu(II)/ascorbate/H2O2 treatment, the abasic site frequency was un-changed, breaks were 0.08/kb, and modified bases were 0.26/kb. Mostor all of the induced breaks have ligatable 5* phosphoryl ends (9).Certain G / C sequence motifs are especially prone to in vitro basemodifications at G and C positions (9). The Nth/ Fpg lanes representLMPCR signal from breaks / modified base / abasic sites. Control

FIG. 2. (a) H2O2-induced footprints in human foreskin fibroblastsexperiments (data not shown) ensured that the observed in vivo dam-along the transcribed strand of the PGK1 promoter from 5*-G0165age patterns were not caused by post-DNA extraction artifacts. Theto C0217 (GGGTA CTAGTGAGAC GTGCGGCTTC CGTTTGTCACarrows point to the locations on the autoradiogram of the base posi-GTCCGGCACG CCGC*G*AAC0217) using primer set G (9). Posi-tions denoted.tions G0214 (214 bases upstream of transcription initiation) and C0213

(* bases) show strong positive footprints. These are included in theDNase I footprint which extends from0187 to0223 (bold-type bases)

by Fe(III)/ascorbate/H2O2 [in 0.3 M sucrose-containing (7). The minimal HIF-1 enhancer domain (underlined bases) (19)extends from 0194 to 0211. Cells were treated as described underbuffer (29)]. Consequently, we cannot identify theMaterials and Methods. The arrows point to the locations on thebound redox cycling ligand(s) responsible for the in vivoautoradiogram of the base positions denoted. (b) Repair of HIF-1base damage. footprint. Cells were treated as in (a) except 5 mM H2O2 was used

The in vivo pattern revealed a few deviations from and half of the treated cells were replaced in fresh complete culturemedium for 24 h of repair before DNA extraction.the in vitro pattern (e.g., base T0110 , Fig. 1; bases G0214 ,

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FIG. 3. Position of prominent positive footprints on the transcribed (lower) strand of the PGK1 promoter. The intensity of the footprintsare scaled from / (least intense) to ///// (most intense). The positions afforded protection from DNase I cleavage by binding of thetranscription factors SP1 and HIF-1 are indicated by black bars.

induces a uniform DNA break distribution along naked ping by LMPCR. Therefore, we assessed to what extentartifacts caused by exposure to high concentrationDNA, while bound proteins protect, effecting negative

footprints. The in vivo H2O2-induced footprint illus- H2O2 contributed nonphysiologic distortion of the ob-served damage frequency patterns. Disruption of chro-trated in Fig. 2a represents a completely different

mechanism. It is positive and primarily involves base matin structure was assessed by the following experi-ment: After exposure to 50 mM H2O2 for 30 min atdamage rather than strand breaks.

The prominent positive footprint shown in Fig. 2a 377C in serum-free medium, human fibroblasts wereimmediately exposed to 10,000 J/m2 of ultraviolet Boccurs at positions G0214 and C0213 in the hypoxia-in-

ducible factor 1 (HIF-1) binding site of the transcribed light. The ultraviolet B light-induced cyclopyrimidinedimer frequency pattern (measured by LMPCR as de-strand (Fig. 3), but not the nontranscribed strand (data

not shown). HIF-1 binds the hypoxia-inducible en- scribed in Refs. 7 and 8) induced in vivo in fibroblastsexposed to H2O2 was compared to the pattern inducedhancer sequence of several genes for gluconeogenic and

glycolytic enzymes (e.g., PGK1 and LDH-A) and growth in vivo in control (no H2O2 exposure) fibroblasts and tothe pattern induced in vitro in purified human genomicfactors (e.g., EPO) (19). PGK1 expression is stimulated

2- to 3-fold by a 1% oxygen environment (19); Co(II) DNA. Figure 4 shows the cyclopyrimidine dimer fre-quency pattern determined using primer set A, whichions and the iron chelator desferrioxamine also induce

HIF-1 expression (20). Induction is slow in comparison maps a portion of the transcribed strand (9); these dataare representative of all the primer sets tested. Theto our 30-min H2O2 treatment. Damage at the two posi-

tions has two discernible components, base damage position of photo-footprints induced in vivo was identi-cal in H2O2-exposed vs control fibroblasts, indicating(57%) and strand breaks (43%). The percentage of

breaks is somewhat greater than the global in vivo or that chromatin structure was not disrupted by expo-sure to 50 mM H2O2.in vitro break frequency of 19% as determined from

analysis of DNA fragment migration on neutral dena- Both the base damage and strand break componentsof the HIF-1 footprint are repaired but at differentturing agarose gels and confirmed in Fig. 1. The in vivo

G0214 signal represents 1/113 molecules with a strand rates. The base damage at positions G0214 and C0213

are 52 and 91% repaired in 24 h, respectively (Fig. 2b),break or oxidized Gua and 1/344 molecules with astrand break. In vivo G0214 shows a 47-fold increase in rates similar to that measured for global base damage

repair. Global single-strand breaks are almost com-base damage and a 10-fold increase in strand breaksrelative to the damage at that position in vitro, as quan- pletely repaired in 30 min (21); however, in the foot-

print, they are repaired about 100 times more slowly,tified with a phosphorimager. The C-213 signal repre-sents 1/322 molecules with a break or oxidized cytosine at 39% repair for position G0214 and 55% repair for

position C0213 in 24 h.and 1/448 molecules with a break. Both the DNase Ifootprint (7) and the H2O2 footprint (data not shown)are absent from the inactive PGK1 allele on the inac-

DISCUSSIONtive X chromosome of females.

Exposure of human male fibroblasts to a concentra- The frequency of in vivo DNA base damage causedby a common endogenous human mutagen, H2O2, hastion of H2O2 several orders of magnitude higher than

those generated in vivo under basal metabolic condi- been mapped. Single-strand breaks with 5* phosphorylends amount to 19% of lesions. The base damage (81%tions was required in order to generate sufficient DNA

base damage for the purpose of damage frequency map- of lesions) patterns found in most regions of the genome

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HOT SPOT FOR HYDROGEN PEROXIDE-INDUCED DNA DAMAGE 211

ments reported elsewhere (29) suggest that H2O2 mayhave released transition metal ions from normally se-questered extranuclear sites, making the availabilityof transition metal ions available for DNA bindinggreater than that expected under basal metabolic con-ditions. Larger nonsequestered transition metal ionpools would most likely facilitate DNA binding at low-affinity binding sites to a greater extent than at high-affinity binding sites, which may become saturatedwith metal ions; therefore, this effect may tend to‘‘smooth out’’ the differences in DNA damage frequencybetween certain nucleotide positions.

A footprint of high damage frequency just outsidethe minimal HIF-1 functional enhancer sequence butwithin the DNase I protected region of the HIF-1 tran-scription factor is extremely sensitive to H2O2 damage.After a 50 mM H2O2 treatment, 1% of cells have damageat one of two base positions (G0214 and C0213) in thisH2O2 footprint. The ratio of base damage to strandbreaks in this footprint more closely resembles thatinduced by DNA-bound transition metal ions, com-pared with loosely bound or free ions (11, 22), which isconsistent with damage at the footprint being causedby a bound redox cycling ligand rather than by freeradicals in solution. Hypotheses to be considered arethat the HIF-1–DNA complex involves a ligand whichredox cycles in the presence of H2O2 or induces a confor-mational change which facilitates DNA oxidation bybound transition metal ion. Recently, Conte et al. (23)have shown that iron ions may replace zinc at zinccoordination positions in DNA-bound zinc finger pro-teins and that such bound iron ions are capable of par-

FIG. 4. Footprint pattern of ultraviolet B light-induced cyclopyri- ticipating in oxidizing reactions which result in site-midine dimers in human foreskin fibroblasts along the transcribed specific DNA damage. The HIF-1 amino acid sequencestrand of the PGK1 promoter using primer set A (9). Cyclopyrimidine has been deduced from a cDNA clone (24). Computerdimer frequency patterns were determined by the LMPCR procedure

analysis does not indicate a consensus transition metaldescribed in Refs. 7 and 8. Lanes 5 and 6 (T) represent the cyclopyri-ion binding sequence, nor are there any consensus zincmidine dimer frequency pattern induced in purified human genomic

DNA in vitro; lanes 7 and 8 (V) represent the cyclopyrimidine dimer finger sequences. However, it does contain a basic he-frequency pattern induced in human fibroblasts in vivo; lanes 9 and lix–loop–helix motif. Certain helix–loop–helix DNA10 (V/H2O2) represent the cyclopyrimidine dimer frequency pattern binding proteins have been shown to induce local DNAinduced in human fibroblasts in vivo after exposure to ultraviolet B

bending (25).light plus 50 mM H2O2. The positions of footprinted bands (bothThe strand breaks at both base positions in this foot-positive and negative) are pointed out by the arrows to the left of

the autoradiogram. The minimal enhancer domains and the DNA- print are unusually refractory to repair and wouldbinding proteins assigned to them are denoted to the right of the probably induce a chromosome break or sister chroma-autoradiogram. tid exchange if replicated under this condition. This is

consistent with the clastogenic activity of H2O2 (26).After damage has occurred, either HIF-1 remainsbound making the breaks inaccessible to repair or asurveyed to date (this report and 29) are identical to

those induced in vitro by redox cycling of bound transi- substantial proportion of breaks at those positions ac-tually are present as a break and base damage doubletion metal ions. These data indicate that the principal

determinants of H2O2-induced DNA damage in vivo are lesion which could be poorly recognized by repair sys-tems. There is precedent for this model considering therelated to the primary DNA sequence.

Exposure of cultured human fibroblasts to cytotoxic iron-binding oligopeptide antitumor agent bleomycin.This agent binds DNA, bringing bound redox cyclingconcentrations of H2O2 was required to generate suffi-

cient DNA damage for damage frequency mapping by ferrous ion in close proximity to DNA. Bleomycin-in-duced DNA lesions are often closely opposed (27);LMPCR. We have shown that chromatin structure re-

mains intact despite this exposure. However, experi- closely opposed lesions are resistant to certain repair

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RODRIGUEZ ET AL.212

12. Pfeifer, G. P., and Riggs, A. D. (1993) Methods Mol. Biol. 23,endonucleases (27). Additionally, the break frequency169–181.at the footprint is almost certainly well above that re-

13. Aruoma, O. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M.quired to saturate the rate-limiting repair step(s) (28); (1991) Biochem. J. 273, 601–604.thus, break repair at the footprint is expected for ki- 14. Boiteux, S., Gajewski, E., Laval, J., and Dizdaroglu, M. (1992)netic reasons to be slow relative to the global repair Biochemistry 31, 106–110.rate. 15. Dizdaroglu, M., Laval, J., and Boiteux, S. (1993) Biochemistry

32, 12105–12111.16. Hatahet, Z., Kow, Y. W., Purnal, A. A., Cunningham, R. P., and

ACKNOWLEDGMENT Wallace, S. S. (1994) J. Biol. Chem. 269, 18814–18820.17. Bryan, S. E., Vizard, D. L., Beary, D. A., LaBiche, R. A., andWe thank Mr. Steven Bates for providing cultured cells.

Hardy, K. J. (1981) Nucleic Acids Res. 9, 5811–5823.18. Tullius, T. D., and Dombroski, B. A. (1986) Proc. Natl. Acad. Sci.

REFERENCES USA 83, 5469–5473.19. Firth, J. D., Ebert, B. L., Pugh, C. W., and Ratcliffe, P. J. (1994)1. Ames, B. N., and Gold, L. S. (1991) Mutat. Res. 250, 3–16.

Proc. Natl. Acad. Sci. USA 91, 6496–6500.2. Holms, G. E., Bernstein, C., and Bernstein, H. (1992) Mutat. Res. 20. Wang, G. L., and Semenza, G. L. (1995) J. Biol. Chem. 270,

275, 305–315. 1230–1237.3. Oller, A. R., and Thilly, W. G. (1992) J. Mol. Biol. 228, 813–826. 21. Kaminskas, E., and Li, J. C. (1992) Mutat. Res. 274, 103–110.4. Cadenas, E. (1989) Annu. Rev. Biochem. 58, 79–110. 22. Luo, Y., Han, Z., Chin, S. M., and Linn, S. (1994) Proc. Natl.

Acad. Sci. USA 91, 12438–12442.5. Marnett, L. J., and Burcham, P. C. (1993) Chem. Res. Toxicol. 6,23. Conte, D., Narindrasorasak, S., and Sarkar, B. (1996) J. Biol.771–785.

Chem. 271, 5125–5130.6. Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Proc.24. Wang, G. L., Jiang, B-H., Rue, E. A., and Semenza, G. L. (1995)Natl. Acad. Sci. USA 90, 7915–7922.

Proc. Natl. Acad. Sci. USA 92, 5510–5514.7. Pfeifer, G. P., and Riggs, A. D. (1991) Genes Dev. 5, 1102–1113.25. Harrington, R. E., and Winicov, I. (1994) Prog. Nucleic Acids

8. Pfeifer, G. P., Drouin, R., Riggs, A. D., and Holmquist, G. P. Res. Mol. Biol. 47, 195–270.(1991) Mol. Cell. Biol. 12, 1798–1804. 26. Oya, Y., Yamamoto, K., and Tonomura, A. (1986) Mutat. Res.

9. Rodriguez, H., Drouin, R., Holmquist, G. P., O’Connor, T. R., 172, 245–253.Boiteux, S., Laval, J., Doroshow, J. H., and Akman, S. A. (1995) 27. Povirk, L. F., and Houlgrave, C. W. (1987) Biochemistry 27,J. Biol. Chem. 270, 17633–17640. 3850–3857.

10. Boiteux, S. (1993) J. Photochem. Photobiol. B 19, 87–96. 28. Kaufmann, W. K., and Wilson, S. J. (1990) Mutat. Res. 236, 107–117.11. Drouin, R., Rodriguez, H., Gao, S., Gebreyes, Z., O’Connor, T. R.,

Holmquist, G. P., and Akman, S. A. (1996) Free Radical Biol. 29. Rodriguez, H., Holmquist, G. P., D’Agostino, R., and Akman,S. A. (1997) Cancer Res., In Press.Med., 21, 261–273.

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