Proc. Nati. Acad. Sci. USA Vol. 82, pp. 8354-8358, December 1985 Biochemistry III recognizes urea residues in oxidized DNA (DNA repair/oxidative stress/hydrogen peroidde//thymine damage) YOKE WAH KoW AND SUSAN S. WALLACE Department of Microbiology, New York Medical College, Valhalla, NY 10595 Communicated by Bruce Ames, August 9, 1985

ABSTRACT exonuclease Im was found to well as from P-L Biochemicals. III was puri- be associated with an activity that recognizes urea residues in fied as described (13). DNA but not thymine glycol residues from which the urea Nucleic Acids and Derivatives. PM2 [3H]DNA was prepared residues were prepared. This activity was not due to a as described (12) and was always >95% form I. Poly([2- contaminating activity such as endonuclease m since urea- '4C]dT) was prepared according to Breimer and Lindahl (14) containing DNA was a competitive inhibitor of exonuclease III with the following modifications. The reaction mixture (1 ml) when apurinic DNA was used as a substrate and vice versa. The contained 40mM potassium cacodylate (pH 6.8), 40mM KCl, apparent kinetic constants for both the substrate and inhibitor 1 mM CoCl2, 1 mM dTTP, 2 ACi of [2-14C]dTTP (45 were determined. Like its apurinic activity, exonuclease mI mCi/mmol; 1 Ci = 37 GBq), 5 AtM oligo(dT)4, and 180 units activity against urea residues was endonucleolytic, nicking on of terminal deoxynucleotidyl . After 8 hr at 350C, the 5' side of the damage and having an optimal Mg2" the reaction was stopped with 200 ,Al of 3 M sodium acetate concentration between 2 and 10 mM. Also, the recog- and 3 ml ofcold ethanol. The newly synthesized poly(dT) was nized alkali-stable damages produced in DNA by H202 in vitro. allowed to precipitate overnight at -70'C. Poly(dT) was We suggest that it may be this activity of exonuclease III that collected by centrifugation at 10,000 rpm (Sorvall SS-34) for accounts for its biological role in vivo. 30 min and washed once with cold 75% ethanol. Poly(dT) was then redissolved in 1 ml of 10 mM Tris HCl, pH 7.5/1 mM Exonuclease III ofEscherichia coli is a complex enzyme that EDTA. This procedure yielded at least 70% ofthe input dTTP contains 3' exonuclease and activities specific (as based on either the radioactive count or the absorbance for duplex DNA (1, 2) as well as an apurinic (AP) endonu- at 260 nm). Unlabeled poly(dA) was purchased from P-L cleolytic activity (3-5). Even though the AP activity of Biochemicals. [methyl-3H]dTTP and [2-14C]dTTP were pur- exonuclease III accounts for about 90% of the total AP chased from Schwarz/Mann. activity in the E. coli cell (6, 7), xth mutants that lack Other Reagents. Agarose, urea, and hydroxyapatite were exonuclease III (8) are not markedly sensitive to alkylating purchased from Bio-Rad; Polygram SIL G/UV254 and agents, such as methyl methanesulfonate, that produce AP Polygram Cell 300 UV254 were from Brinkmann Instruments; sites (6, 7). Further, single-strand breaks produced by ion- Sephadex G-75 was from Pharmacia Fine Chemicals; H202 izing radiation may contain 3' phosphate termini or 3' glycolic (30%) was from Fisher Chemicals; and OS04 was from ends (9). Such termini would require a 3' phosphatase- Polyscience (Warrington, PA). exonuclease activity so that the strand breaks would be a Preparation of Substrates. For preparation of thymine substrate for repair by DNA polymerase I. Although glycol-containing PM2 DNA, PM2 DNA was preheated at exonuclease III accounts for >99% of this activity in E. coli 650C for 2-5 min and then brought to 0.04% OS04 from a 4% (10), xth mutants are not sensitive to x-rays (7). stock. The DNA was incubated for another 5 min, quickly The most profound phenotype thus far associated with cooled on ice, and then dialyzed extensively to remove the exonuclease III deficiency is the extreme sensitivity of xth OSO4. The treatment produced approximately one thymine mutants to H202, about 20-fold greater than that ofwild type glycol per DNA molecule. The number ofthymine glycols per (11). We report here that exonuclease III can endonucleo- DNA molecule was determined after exhaustive digestion lytically incise adjacent to alkali-stable fragmented residues with endonuclease III (13). The number of endonuclease ofthymine in DNA, and we propose that this activity may be III-sensitive sites produced under these conditions is about responsible for repairing DNA damage produced by free half the number of 5,6-dihydroxydihydrothymine-type dam- radicals generated by metabolic oxygen and that this may be ages, as determined by the acetol fragment assay (13), and is the principal biological role for exonuclease III. equal to the number of anti-thymine glycol antibody sites (unpublished data). To produce greater numbers of thymine glycols per DNA molecule, proportionally higher concentra- MATERIALS AND METHODS tions ofOS04 were used for oxidation. For preparing thymine Bacteria and Bacteriophage. E. coli AB3027, a mutant in glycol-containing poly([2-14C]dT), the same procedure was exonuclease III, was obtained from the E. coli Genetic Stock used except that the OS04 concentration was brought to 2%. Center (Yale University) and used for the preparation of The treated polynucleotide was quickly chilled on ice and endonuclease III. PM2 bacteriophage and host Altermonas OS04 was then removed by ether extraction followed by espejiana were grown as described (12). exhaustive dialysis. . DNA polymerase I (E. colt) Klenow fragment For preparation of urea-containing PM2 DNA or poly([2- was purchased from P-L Biochemicals, and exonuclease III, 14C]dT), thymine glycol-containing nucleic acids were sub- a physically homogeneous enzyme prepared from E. coli jected to alkaline hydrolysis at room temperature by dialyz- K12BE257/pSGR3, a thermoinducible overproducer of ing against 40mM KH2PO4, pH 12.0/2 mM EDTA overnight. exonuclease III, was obtained from New England Biolabs as Alkaline hydrolysis converts the thymine glycol residues on DNA to urea and N-fragmented residues of thymine. Alka- line-hydrolyzed nucleic acids were then dialyzed against 10 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: AP, apurinic. 8354 Downloaded by guest on September 30, 2021 Biochemistry: Kow and WaHace Proc. Natl. Acad. Sci. USA 82 (1985) 8355

mM Tris HCl, pH 7.5/1 mM EDTA. The number of urea- or immediately placed on glass fiber filters (GF/A), and washed N-fragmented thymine residues formed by this process was with ice-cold 10% trichloroacetic acid followed by washing equivalent to the parental number of thymine glycols (see with ice-cold 95% ethanol. The reaction mixtures were then Table 1). No alkali-labile sites were generated either during incubated at 370C and 25-gl samples were taken every 20 min the oxidation of thymines to thymine glycol or during the and treated as above. All filters were dried and assayed for alkali cleavage of thymine glycols to urea. radioactivity. For preparation of AP DNA, PM2 DNA was precipitated Analysis for the Enzymatic Release of Urea from Urea- with ethanol, resuspended in 10 mM sodium citrate/100 mM Containing poly([2-14C]dT)-poly(dA). Urea-containing NaCl, pH 5.0, and heated at 70'C for 7 min (15). This poly([2-14C]dT) was heated to 70'C and an equimolar amount treatment produced approximately one AP site per DNA of poly(dA) was added. The mixture was then allowed to molecule. Acid/heat-treated DNA was quickly cooled on ice, anneal slowly. The double-stranded polynucleotide was then precipitated with ethanol, and resuspended in 10 mM digested exhaustively with excess endonuclease III or Tris HCl, pH 7.5/1 mM EDTA. The number of AP sites was exonuclease III for 6 hr, evaporated to dryness under vacuum determined by fluorometry after alkali hydrolysis (16). at room temperature, and then resuspended in 50 j1. of H20. Endonuclease Assays. Endonuclease activity of endonucle- The total volume was then spotted onto a silica TLC plate and ase III and exonuclease III was determined by fluorometry or developed with a mixture of ethyl acetate/2-propanol/H20 agarose gel electrophoresis. For thymine glycol- or urea- (75:16:9). Authentic markers were developed simultaneously containing PM2 DNA, the activity was estimated either by and visualized as described (13). fluorometry or gel electrophoresis, whereas for AP DNA, Inhibition Studies. PM2 [3H]DNA containing urea residues due to the alkaline instability of AP sites, the enzyme activity was allowed to compete with various amounts of unlabeled was determined by gel electrophoresis. For endonuclease III, AP PM2 DNA. Time points were taken so that initial velocity 200 ,ul of reaction volume contained 10 mM Tris HCl (pH of the reaction could be followed. Similar experiments were 7.5), 1 mM EDTA, 0.1 M KCl (13), and 200-400 ng of DNA. carried out with labeled AP PM2 DNA that was allowed to To this, 20 ,ul of diluted enzyme was added and the reaction compete with unlabeled urea-containing DNA. Each time mixture was incubated at 37°C for 10 min. For exonuclease point was analyzed by agarose gel electrophoresis to deter- III, 200 ,u1 of reaction volume contained 66 mM Tris HCl (pH mine the rate of the reaction. 8.0), 2.0 mM MgCl2, 1 mM mercaptoethanol (17), and H202 Treatment of PM2 DNA. PM2 DNA was treated with 200-400 ng of DNA. Diluted enzyme, 20 A.l, was added and 250 ,uM FeSO4/125 ;kM EDTA and H202 from 0.1 to 10 mM the reaction mixture was incubated at 37°C for 5 min. One at 4°C for 10 min. The FeSO4/EDTA and H202 were removed unit of enzyme activity was defined as the amount of enzyme rapidly from the DNA by a minicolumn prepared with a required to yield 1 nmol of nicks per min of reaction time at Microfuge tube. A Microfuge tube was packed with 1.2 ml of 370C. swollen Sephadex G-75 and the column was equilibrated by For the fluorometric assay, the enzyme reaction was washing three to five times with 10 mM Tris HCl, pH 7.5/1 terminated with 1.5 ml of a solution containing 20 mM mM EDTA. Treated DNA, 200 /ul, was layered on top of the KH2PO4 (pH 11.8), 2 mM EDTA, and 0.5 ,ug of ethidium column and centrifuged for 75 sec. About 270 ,ul of eluate was bromide per ml. The average number of enzyme-sensitive collected and the recovery of DNA ranged from 50% to 80%. sites was estimated as described (16). The H202-treated DNA was then allowed to react with either For agarose gel electrophoresis, 40 ,lI (one-fifth the volume exonuclease III or endonuclease III, and the number of of the reaction medium) of a solution was added that alkali-stable sites and single-strand breaks was determined by contained 30% glycerol, 0.025% bromophenol blue, 0.1 M both fluorometry and gel electrophoresis. KH2PO4, 10 mM EDTA, and 1 M KCl (pH 10.0). At pH 10.0, AP sites were not cleaved within the experimental period. RESULTS Samples, 20 ,4, were loaded onto a 0.8% agarose gel and electrophoresed at 100 V for 4 hr. DNA bands were stained PM2 DNA Containing Urea and N-Fragmented Thymine with 0.5 ,g of ethidium bromide per ml. For unlabeled PM2 Residues Is a Substrate for Exonuclease mII. PM2 DNA was DNA, the amount of form I and form II was determined by oxidized with OS04 to produce thymine glycols and then scanning densitometry (Biomed Instruments, model SL- digested exhaustively with alkali to cleave the glycols, TRFF); for labeled PM2 DNA, DNA bands were cut, leaving urea and N-substituted urea fragments (13, 18). Table dissolved in 2 ml of 1 M HCl, and assayed for radioactivity 1 shows that thymine glycol-containing DNA was a substrate in a scintillation counter. The average number of enzyme- for endonuclease III (13) but not exonuclease III, whereas the sensitive sites was then determined by the formula: nicks/DNA = -ln(fraction of form I remaining). In cases in which the number of damages was quantitated in DNA (Table Table 1. Recognition by exonuclease III of urea or N-fragmented 1), the reaction was run to completion. residues of thymine Determination of the Nature of Exonuclease m Nicks on Alkali-stable, enzyme-sensitive sites Urea-Containing DNA. PM2 DNA, 400 ng, untreated or per PM2 DNA molecule, no. urea-containing (about 3-4 sites per molecule), was incubat- III ed for 20 min at 37°C in the respective Exo reaction buffers for and endonuclease III and exonuclease III with or without a PM2 DNA substrate Exo III Endo III endo III 10-fold excess enzyme. The reactions were terminated by heating at 70°C for 5 min and the endonuclease III- and Untreated 0 0 0 exonuclease III-induced nicks were analyzed for their ability OS04 (0.04%) treatment 0 0.85 0.85 to serve as substrates for DNA polymerase I (13). At 4°C, to + Alkali digestion 0.90 0.86 0.89 each reaction mixture was added 20 nmol (each) of dATP, OS04 (0.08%) treatment 0.08 1.62 1.65 dGTP, and dCTP as well as 0.8 nmol of unlabeled dTTP + Alkali digestion 1.65 1.65 1.70 together with 2.5 ,uCi of [3H]dTTP. The reaction volume was OS04 (0.16%) treatment 0.12 3.5 3.6 brought to 200 /l. with 25 mM Tris HCl (pH 7.5), and the + Alkali digestion 3.6 3.6 3.6 MgCl2 concentration was adjusted to 8 mM. To this was Exo III, exonuclease III. Endo III, endonuclease III. The number added 1 unit of DNA polymerase I. After addition of DNA of sites per molecule >2 cannot be accurately quantitated assuming polymerase 1, 25 ,ul was removed from each reaction mixture, a Poisson distribution. Downloaded by guest on September 30, 2021 8356 Biochemistry: Kow and Wallace Proc. Natl. Acad Sci. USA 82 (1985)

A 4U 2 and 12 mM MgCl2 (data not shown). A similar Mg2' requirement was observed when AP DNA was used as the 3 substrate (17). .9 To determine the nature of the exonuclease III-induced nick on urea-containing DNA, a DNA polymerase suscepti- E2 bility assay (17) was used. Fig. 2 shows that after treatment with exonuclease III for 20 min, urea-containing DNA -I -0.5 0.5 ,- became a good substrate for DNA polymerase I; however, E ue N)n this same urea-containing DNA after treatment with endo- B~~~~~ III was not a good substrate for DNA polymerase I. Endonuclease III has been shown to nick on the 3' side of thymine glycols (13, 19) and the same appears to be true for urea-containing DNA. However, exonuclease III appeared to nick on the 5' side of the urea residue in similar manner to its action on AP DNA (17). The argument could be made that exonuclease III nicked on the 3' side of the urea residue and then exonucleolytically cleaved the urea monophosphate, leaving a substrate for DNA polymerase I. We think that this is unlikely since we did not detect any released urea mononucleotide by TLC analysis after digestion ofpoly(dA)-

-2 -I 2 poly(dT)-containing urea residues (see next section). CAP DNA] nM-' Exonuclease m Recognizes Urea as an Endonuclease Not a Glycosylase. Urea-containing poly([2-14C]dT) annealed to FIG. 1. Inhibition studies with alternative substrates for poly(dA) was digested exhaustively with either exonuclease exonuclease III. Either AP DNA was used as substrate and urea- III or endonuclease III. Fig. 3 shows a thin-layer chromato- containing DNA (urea DNA) as inhibitor (A) or urea DNA was used gram was as substrate and AP DNA as inhibitor (B). PM2 [3H]DNA (3 X 104 ofthe products ofthis digestion. Urea released only cpm/,g of DNA) was used as substrate, whereas unlabeled DNA after digestion with endonuclease III, which recognizes this was used as inhibitor. (A) AP DNA contained 1.8 AP sites per DNA product as a glycosylase (13). No urea was released with molecule, whereas urea DNA contained 2.7 enzyme-sensitive sites exonuclease III digestion, supporting the idea that the per DNA molecule. For each 200-,ul reaction, 0.054 Weiss unit exonuclease III behaves as an endonuclease not a glycos- (equivalent to 0.036 ,g of protein) was used. One Weiss unit is ylase nicking on the 5' side of the urea residue. defined as the amount of enzyme required to release 1 nmol of Reaction of Exonuclease m with H202-Treated DNA. Since acid-soluble radioactivity in 30 min at 37°C. (B) AP DNA contained mutants defective in exonuclease III (xth) are very sensitive per and urea DNA 2.1 AP sites DNA molecule contained 2.9 to H202 (11), we attempted to generate alkali-stable, enzyme-sensitive sites per DNA molecule. Inhibitor concentrations exonuclease sites in DNA with H202 in were 0 nM (o), 0.4 nM (v), 1.0 nM (A), and 2.0 nM (a). III-sensitive treated vitro. Since treatment with 0.2 M H202 in Tris HCl, pH 7.5/1 mM EDTA primarily produced strand breaks and very few urea-containing DNA was a substrate for both enzymes. alkali-stable, endonuclease III- and exonuclease III-sensitive Further, the number of urea residues recognized by the sites (data not shown), we decided to treat the DNA with exonuclease III was equivalent to the number of parental H202 in a Fenton-type reaction that generates OH radical thymine glycols, as assayed by the endonuclease III. The species (20). Fig. 4 shows that when DNA was treated with exonuclease III activity on urea-containing DNA was not due a mixture of H202 and FeSO4/EDTA complex, both alkali- to an endonuclease III contaminant since endonuclease III stable, endonuclease III and exonuclease III sites were recognized both thymine glycol- and urea-containing DNA, generated among a background of single-strand breaks. whereas exonuclease III only recognized urea fragments but Demple and Linn also showed that H202 treatment of DNA not thymine glycols. results in the production of endonuclease III-sensitive sites To further substantiate that the recognition of urea frag- (21). These data suggest that the alkali-stable sites generated ments by exonuclease III was not due to a possible contam- by the Fenton reaction under these conditions are a mixture inant that might only recognize urea but not thymine glycol, of thymine glycols with urea and possibly formylurea and urea-containing DNA was tested for its ability to compete formamide. The latter are structurally similar to urea and the with exonuclease III activity on AP DNA. Fig. 1 shows that fragments might be small enough to provide the necessary urea-containing DNA was a competitive inhibitor ofAP DNA space for exonuclease III recognition and incision. and vice versa. The kinetic constants obtained from these Attempts to determine whether exonuclease III recognized data are given in Table 2. The fact that exonuclease III other alkali-stable residues in damaged DNA were not activity on AP or urea-containing DNA was competitively successful. Potassium permanganate (0.1 mM) treatment of inhibited by the alternative substrate supports the idea that it PM2 DNA at pH 7.5 for 20 min at room temperature yielded is exonuclease III that is recognizing the urea fragment. about two endonuclease III-sensitive sites; however, no Reaction Conditions and Nature of the Nick. We found that exonuclease III-sensitive sites were generated even after the exonuclease III-induced nicking of urea-containing PM2 preincubation of the permanganate-treated DNA at 370C for DNA was dependent on Mg2", with optimal activity between 4 hr prior to exonuclease III digestion. Presumably under Table 2. Kinetic parameters of exonuclease III activity on AP and urea-containing DNA Substrate Inhibitor KPVP ,* mM kPP AP DNA Urea-containing DNA 3.5 mM* (0.63 nM)t 5.1 x 104:' 6.8 mM* (2.76 nM)§ Urea-containing DNA AP DNA 9.5 mM* (1.84 nM)§ 7.5 x 10t 2.0 mM* (0.42 nM)t *mM . tnM AP sites. §nM urea-containing residues. fnmol of "nicks" per min per mg of protein. Downloaded by guest on September 30, 2021 Biochemistry: Kow and WaHace Proc. Natl. Acad. Sci. USA 82 (1985) 8357

-5 16 *\ 4 v z c0v 2~ 8~~~~~ a- 0 !X N 08h 10 10 30 4 C',.I4 c 0.4 6 0-~~~~~ Z 0,0 E 0--eo ----_o c 2 3 4 -l og I H2023 10 20 30 40 Reaction Time (min) FIG. 4. Formation of enzyme-sensitive sites and strand breaks in H202-treated PM2 DNA. H202 at the indicated concentrations was FIG. 2. Exonuclease III-induced nicks as substrates for DNA incubated with PM2 DNA for 3 min at 4°C in the presence of 250 ,uM polymerase I. Urea-containing PM2 DNA was incubated with FeSO4/125 ,AM EDTA. H202 and Fe2+ were removed rapidly by a endonuclease III (e), exonuclease III (o), endonuclease III/exonu- minicolumn method. Single-strand breaks (e), alkali-stable, endo- clease III (A), and exonuclease III/endonuclease III (A). After the nuclease III-sensitive sites (A), and alkali-stable, exonuclease III- reactions were terminated, 1 unit of DNA polymerase, 20 nmol sensitive sites (o) were determined by fluorometry and electro- (each) of dATP, dGTP, and dCTP, 0.8 nmol of dTTP, and 3 ,Ci of phoresis. [3H]dTTP were added, and the reaction mixtures were incubated at 370C for the indicated intervals. "space" is necessary for substrate recognition by exonucle- ase III. these conditions, the DNA should have contained thymine glycols and methyltartronylurea residues (22). Attempts to DISCUSSION produce formylurea residues by periodate oxidation of thymine glycol were also unsuccessful due to the substantial Compatibility with Proposed Reaction Mechanism. Exonu- number ofsingle-strand breaks generated. However, the data clease III, a monomeric protein having a molecular weight of with permanganate suggest that methyltartronylurea, due to about 28,000, has been shown to have at least four catalytic its much longer length, is not a substrate for exonuclease III, activities (23): (i) a 3' exonuclease activity, which hydrolyzes although it is for endonuclease III, supporting the idea that a the 3' terminal phosphodiester bond of DNA; (ii) a 3' H activity, which hydrolyzes the 3' phosphodiester bond of the RNA strand in a DNA-RNA Urek Thymine T BA hybrid; (iii) a 3' phosphatase activity, which hydrolyzes the Glycol 3' terminal phosphomonoester of DNA; and (iv) an AP or apyrimidinic endonuclease activity, which hydrolyzes the phosphodiester bond of DNA at the AP sites. Since 3000 exonuclease III is a small protein, Weiss (23) proposed a common-site model to explain all of these reactions. The model requires the enzyme to have three regions of recog- nition: (i) a region that recognizes the phosphoester bond and is responsible for the cleavage-that is, the ; (ii) a E 0a 2000O region that recognizes the need for a duplex, either an u RNA-DNA or DNA-DNA duplex; and (iii) a region that recognizes the space created either by the loss of a base (AP site) or a breathing 3' end. Based on this model and comparing the similarity of urea residues on DNA to AP sites, we thought that the short urea I000F chain might provide enough space for recognition by exonuclease III. Table 1 illustrates that exonuclease III recognized the alkali hydrolysis product(s) of thymine glycol residues on DNA that have been shown to contain urea residues (13, 18). Studies in this laboratory with model 0 5 10 compounds (unpublished data) have shown that alkali hy- Distance Migrated(cm) drolysis of thymine glycol yielded mostly urea; thymidine glycol yielded two isomers of urea deoxyribose, the FIG. 3. Analysis of products released from urea-containing pyranoside and the furanoside; whereas thymidine glycol poly([2-14C]dT) after treatment with exonuclease III and endonucle- monophosphate yielded primarily deoxyribosylurea mono- ase III. Thymine glycol-containing poly([2-14C]dT) was prepared by phosphate. Furthermore, the quantitative conversion of oxidizing poly([2-'4C]dT) with 2% OS04 for 30 min at 65°C, quickly thymine glycol residues to exonuclease III-sensitive, alkali- cooled, and dialyzed extensively in 10 mM Tris HCl, pH 7.5/1 mM stable sites supports the idea that the alkali-digested DNA EDTA. Thymine glycol residues on poly([2-14C]dT) were converted contained mostly urea residues and that exonuclease III to urea residues by alkali hydrolysis. Urea-containing poly([2- recognized these residues on DNA. '4C]dT) was annealed to poly(dA) by heating equimolar amounts of both homopolymers to 70°C for 3 min and letting the mixture cool The reaction catalyzed by exonuclease III using urea- slowly to room temperature. Urea-containing poly([2-14C]dT)*poly- containing DNA as a substrate resembled that with an AP (dA) duplex was then incubated at 37°C for 8 hr with exonuclease III DNA substrate with respect to both reaction conditions and (o), with endonuclease III (A), or without enzymes (e). Products site ofthe nick. The enzyme acted as an endonuclease at urea released were then analyzed by TLC. T, thymine; BA, 5-hydroxy- residues in DNA rather than as a glycosylase (Fig. 3), thus 5-methylbarbituric acid. distinguishing it from urea glycosylases isolated from E. coli Downloaded by guest on September 30, 2021 8358 Biochemistry: Kow and Wallace Proc. Natl. Acad Sci. USA 82 (1985) (13, 14) or mammalian cells (24). Further, the competition We are grateful to Dr. Robert Melamede and Michael Laspia for studies (Fig. 1) substantiated that the activity against urea valuable discussions and to Dr. Hiroshi Ide for communicating his residues was catalyzed by exonuclease III and not a con- unpublished data on the formation of urea from thymine glycol. This taminant in the preparation. research was supported by Grant CA33657 awarded by the National Possible Biological Role of Exonuclease m. The specific Cancer Institute. activity of purified endonuclease III (at least 75% pure) 1. Richardson, C. C. & Kornberg, A. (1964) J. Biol. Chem. 239, against urea residues is between 60 and 150 nmol of nicks per 242-250. min/mg of protein (unpublished data; ref. 25), whereas for 2. Richardson, C. C., Lehman, I. R. & Kornberg, A. (1964) J. exonuclease III, the specific activity against urea residues Biol. Chem. 239, 251-258. was 75 umol of nicks per min/mg of protein (Table 2). 3. Hadi, S.-M. & Goldthwait, D. A. (1971) Biochemistry 10, Therefore, the specific activity of exonuclease III is 103-fold 4986-4993. higher than that of endonuclease III. Furthermore, it has 4. Verly, W. G. & Rassart, E. (1975) J. Biol. Chem. 250, been estimated that E. coli contains about 300-400 molecules 8214-8219. III data; 25), and 3500 5. Weiss, B. (1976) J. Biol. Chem. 251, 1896-1901. of endonuclease (unpublished ref. 6. Ljungquist, S., Lindahl, T. & Howard-Flanders, P. (1976) J. molecules of exonuclease III per cell (23). Though the Km for Bacteriol. 126, 646-653. urea-containing DNA is about the same for both enzymes, 7. Yajko, D. M. & Weiss, B. (1975) Proc. Natl. Acad. Sci. USA 1.84 nM ofurea residues for exonuclease III (Table 2) and 1.0 72, 688-692. nM of urea residues for endonuclease III (unpublished data), 8. White, B. J., Hochhauser, S. J., Cintron, N. M. & Weiss, B. the total activity of endonuclease III against urea residues is (1976) J. Bacteriol. 126, 1082-1088. <1% that of exonuclease III. Furthermore, exonuclease III 9. Henner, W. D., Grunberger, S. M. & Haseltine, W. A. (1982) incised on the 5' side of the urea residue (class II AP J. Biol. Chem. 257, 11750-11754. endonuclease), whereas endonuclease III cleaved on the 3' 10. Milcarek, C. & Weiss, B. (1972) J. Mol. Biol. 68, 303-318. 11. Demple, B., Halbrook, J. & Linn, S. (1983) J. Bacteriol. 153, side of the urea residue (class I AP endonuclease), which 1079-1082. does not leave a good substrate for the polymerase I reaction. 12. Wallace, S. S., Katcher, H. L. & Armel, P. R. (1981) in DNA These observations suggest that exonuclease III may be the Repair: A Laboratory Manual of Research Procedures, eds. major repair enzyme in E. coli for urea damages as well as for Friedberg, E. & Hanawalt, P. (Dekker, New York), Vol. 1A, AP sites. pp. 113-125. Exonuclease III mutants (xth) are hypersensitive to killing 13. Katcher, H. L. & Wallace, S. S. (1983) Biochemistry 22, by H202 (11) as well as sensitive to killing by near-UV light 4071-4081. 14. Breimer, L. & Lindahl, T. (1980) Nucleic Acids Res. 8, (300-400 nM) (26). Xth mutants, however, are only slightly 6199-6211. sensitive to alkylating agents, which produce substantial 15. Lindahl, T. & Andersson, A. (1972) Biochemistry 11, numbers of AP sites. This suggests that some type of 3618-3623. alkali-stable base damage(s) is produced by H202 as well as 16. Kowalski, D. (1979) Anal. Biochem. 93, 346-354. by the photodynamic action of near-UV light that can be 17. Gossard, F. & Verly, W. G. (1978) Eur. J. Biochem. 82, repaired by exonuclease III. Given the data presented here, 321-332. 18. Roti Roti, J. L. & Cerutti, P. A. (1974) Int. J. Radiat. Biol. 25, it is conceivable that one of these damages might be urea. 413-417. H202 treatment of PM2 DNA in the presence of Fe2+ 19. Warner, H. R., Demple, B. F., Deutsch, W. A., Kane, C. M. produced exonuclease III-sensitive, alkali-stable sites (Fig. & Linn, S. (1980) Proc. Natl. Acad. Sci. USA 77, 4602-4606. 4). Further, Preiss and Zilling (27) showed that the end 20. MelloFilho, A. C. & Meneghini, R. (1984) Biochim. Biophys. products of ring opening of thymine, thymidine, and thymi- Acta 781, 56-63. in vitro are 21. Demple, B. & Linn, S. (1982) Nucleic Acids Res. 10, dylic acid by 3 M H202 urea, deoxyribosylurea, 3781-3789. and phosphodeoxyribosylurea, respectively. Although these 22. Iida, S. & Hayatsu, H. (1970) Biochim. Biophys. Acta 213, authors used fairly drastic conditions, it is possible that urea 1-13. might be a primary end product ofH202 degradation of DNA 23. Weiss, B. (1981) in The Enzymes, ed. Boyer, P. D. (Academic, in vivo. Massie et al. (28) have shown that base destruction New York), Vol. 14, pp. 203-231. appears to be the most important event in H202-treated 24. Breimer, L. H. (1983) Biochemistry 22, 4192-4197. that near-UV 25. Breimer, L. H. & Lindahl, T. (1984) J. Biol. Chem. 259, DNA. Also, Wang et al. (29) demonstrated 5543-5548. irradiation generates H202 in cell culture medium and Sam- 26. Sammartano, L. J. & Tuveson, R. W. (1983) J. Bacteriol. 156, martano and Tuveson (30) showed that exogenously added 904-906. catalase to near-UV-treated E. coli reduced its lethal and 27. Preiss, H. & Zilling, W. (1965) Physiol. Chem. 342, 73-78. mutagenic effects, suggesting that these effects were due to 28. Massie, H. R., Samis, H. V. & Baird, M. B. (1972) Biochim. H202. Biophys. Acta 272, 539-548. to measure the amount of urea 29. Wang, R. J., Ananthaswamy, H. N., Nixon, B. T., Hartman, Sensitive assay procedures P. D. & Eisenstark, A. (1980) Radiat. Res. 82, 269-276. formed in cells under oxidative stress await development. 30. Sammartano, L. J. & Tuveson, R. W. (1984) Photochem. The formation of urea might also prove to be a marker for Photobiol. 40, 607-612. oxidative damage, as has been shown for thymine glycol by 31. Cathcart, R., Schwiers, E., Saul, R. L. & Ames, B. N. (1984) Cathcart et al. (31). Proc. Natl. Acad. Sci. USA 81, 5633-5637. Downloaded by guest on September 30, 2021