Proc. Nati. Acad. Sci. USAVol. 84, pp. 8917-8921, December 1987Biochemistry

Spontaneous mutagenesis and oxidative damage to DNA inSalmonella typhimurium

(oxyR/catalase/T A to A-T transversions/apurinic sites)

GISELA STORZ, MICHAEL F. CHRISTMAN*, HELMUT SIESt, AND BRUCE N. AMEStDepartment of Biochemistry, University of California, Berkeley, CA 94720

Contributed by Bruce N. Ames, September 2, 1987

ABSTRACT Salmonella typhimurium strains containingdeletions of oxyR, a positive regulator of defenses againstoxidative stress, show 10- to 55-fold higher frequencies ofspontaneous mutagenesis compared to otherwise isogenic oxyR+control strains. The high spontaneous-mutation frequency inoxyR deletion strains is decreased by a factor of 3 when thestrains are grown anaerobically. oxyR deletion strains show anincrease in small deletion mutations and at least three of the sixpossible base-substitution mutations (TA to A-T, COG to T-A,and C-G to A-T). However, the largest increase in mutationfrequency is observed for T-A to A-T transversions (40- to146-fold), the base-substitution mutation most frequentlycaused by chemical oxidants. The introduction into oxyRdeletion strains of multicopy plasmids carrying the oxyR-regulated genes for catalase (kaIG) or alkyl hydroperoxidereductase (ahp) results in overexpression of the respectiveenzyme activities and decreases the number of spontaneousmutants to wild-type levels. The introduction into oxyR dele-tions of a plasmid carrying the gene for superoxide dismutase(sodA) decreases the mutation frequency by a factor of 5 insome strain backgrounds. Strains that contain a dominant oxyRmutation and overexpress proteins regulated by oxyR showlower spontaneous-mutation frequencies by a factor of 2. Theseresults indicate that oxyR and oxyR-regulated genes play asignificant role in defense against spontaneous oxidative DNAdamage. The role of oxidative damage to DNA in "spontane-ous" mutagenesis is discussed.

Spontaneous mutations could arise by several differentmechanisms. These include depurination, deamination, andother forms of DNA damage and repair, as well as recom-bination and errors in DNA replication (1). Among thepotential sources of DNA damage, active oxygen specieshave become recognized as being of biological importance(2-7). Studies have shown that oxygen metabolism influencesDNA damage in mammals (6, 7) and bacteria. Hartman et al.(8) showed that Salmonella typhimurium strains grown underaerobic conditions had increased frequencies of small dele-tion and frameshift mutations relative to strains grownanaerobically, while the frequency of G-C to AT transitionswas depressed under the aerobic conditions. Recently, Farret al. (9) showed that Escherichia coli mutants that lack boththe manganese- and iron-containing superoxide dismutases(sodA sodB double mutants) have enhanced spontaneous-mutation frequencies compared to wild-type strains duringaerobic growth. In addition, several E. coli DNA repairmutants-recA xth double mutants (10), certain polA mutants(11), and polA recB double mutants (12)-form filaments anddie aerobically, though they grow normally under anaerobicconditions.

We recently have identified and characterized a gene,oxyR, that positively controls a regulon involved in cellulardefenses against oxidative stress in both S. typhimurium andE. coli (13, 14). A dominant mutant, oxyRi, is resistant tohydrogen peroxide and constitutively overexpresses ninehydrogen peroxide-inducible proteins. Activities that areoverexpressed in oxyRi mutants include catalase, Mn-con-taining superoxide dismutase, glutathione reductase, and apreviously unreported alkyl hydroperoxide reductase. Strainswith deletions of the oxyR locus are uninducible for theseproteins and are hypersensitive to killing by oxidants (13, 14).

In this paper we examine the role of the oxyR-controlledregulon in spontaneous mutagenesis. We compare the spon-taneous-mutation frequency in the wild-type (oxyR+) mu-tagenicity tester strains with the frequency in strains con-taining either the dominant oxyRI allele or a deletion of theoxyR locus (oxyRA2). We also characterize the types ofspontaneous mutations occurring in the oxyR mutant strainsand examine the role of specific oxyR-regulated genes inproviding defense against oxidative DNA damage.

MATERIALS AND METHODSBacterial Strains. The genetic backgrounds of the strains

used in this study are listed in Table 1. The oxyRI mutantallele and the oxyRA2 deletion were transduced into thevarious tester-strain backgrounds using P22 HT105/1 int-201phage (16) and the linked transposon zii6J4::TnlO. ahp::TnJOwas also transduced directly into the tester strains, whereasTA4188 was constructed by conjugating the plasmid pKM101and transducing hisG428 linked to zec2::TnlO into katGA2(TA4113). The pACYC184 constructs (pAQ5, pAQ6, pAQ7,and pAQ8) were made by using standard recombinant DNAtechniques. pAQ5 encodes the E. coli oxyR gene (M.F.C.,G.S., and B.N.A., unpublished results) on a 1.5-kilobase (kb)BamHI-EcoRV fragment, pAQ6 encodes the E. coli sodAgene (17) on a 1.0-kb Ava III fragment, pAQ7 encodes the E.coli katG gene (18) on a 3.65-kb HindIII fragment, and pAQ8encodes the S. typhimurium ahp gene (L. Tartaglia andB.N.A., unpublished results) on a 8.8-kb BamHI-Sal Ifragment. All of the plasmids were constructed in E. coli,transformed into S. typhimurium SL4213 (restriction minus,modification plus), and then transduced from SL4213 intoTA4116 and TA4119. The levels of catalase, alkyl hydroper-oxide reductase, and Mn-containing superoxide dismutaseactivity in the plasmid-carrying strains were assayed asdescribed (13).

Spontaneous-Mutation Assays. The histidine (His+)-rever-sion assays were carried out as described by Maron andAmes (19). Cells were grown from frozen permanent cultures

*Present address: The Whitehead Institute for Biomedical Research,Nine Cambridge Center, Cambridge, MA 02142.tOn sabbatical leave from: Department of Physiological Chemistry,University of Dusseldorf, Dusseldorf, F.R.G.fTo whom reprint requests should be addressed.

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Table 1.

Strain*

TA4114TA4115TA4116TA4117TA4118TA4119TA4120TA4121TA4122TA4123TA4124

TA4125

TA4126TA4127TA4128

S. typhimurium strains

GenotypehisG428 zii6l4::TnIOhisG428 zii6J4::TnlO oxyRIhisG428 zii6l4::TnJO oxyRA2hisG428 zii6l4::TnlO/pKM101hisG428 zii6I4::TnlO oxyRl/pKM101hisG428 zii6l4::TnIO oxyRA2/pKM101hisG428 zii6l4::TnlO AuvrBbiochll04hisG428 zii6J4::TnIO AuvrBbiochllO04 oxyRIhisG428 zii6)4::TnJO AuvrBbiochllOO4 oxyRA2hisG428 zii6l4::TnlO AuvrBbiochllO04/pKM101hisG428 zii6l4::TnlO AuvrBbiochllOO4 oxyRIlpKM101

hisG428 zii6J4::TnJO AuvrBbiochllO04 oxyRA2/pKM101

hisG46 zii6J4::TnlO AuvrBbiochll004/pKM101hisG46 zii6J4::TnlO AuvrBbiochllO04 oxyRI/pKM101hisG46 zii6l4::TnJO AuvrBbiochllO04 oxyRA2/pKM101

TA4188 hisG428 zec2: :TnlO AkatG/pKM101TA4189 hisG428 ahp::TnJO/pKM101TA4271 hisG428 zii6J4::TnlO oxyRA2/pACYC184TA4272 hisG428 zii6J4::TnlO oxyRA2/pAQ5TA4273 hisG428 zii6J4::TnJO oxyRA2/pAQ6TA4274 hisG428 zii6J4::TnlO oxyRA2/pAQ7TA4275 hisG428 zii6l4::TnJO oxyRA2/pAQ8TA4276 hisG428 zii6l4::Tn)O oxyRA2/pKM101/pACYC184TA4277 hisG428 zii6I4::TnIO oxyRA2/pKM101/pAQ5TA4278 hisG428 zii6l4::TnlO oxyRA2/pKM101/pAQ6TA4279 hisG428 zii6J4::TnlO oxyRA2/pKM101/pAQ7TA4280 hisG428 zii6J4::TnlO oxyRA2/pKM101/pAQ8DB7155 hisC527 leu414 supBDB7302 hisC527 1eu414 supGSODB7303 hisC527 1eu414 supC80DB7304 hisC527 1eu414 tyrU90 (supM)

*The source of all strains is this paper except for DB7155, DB7302,DB7303, and DB7304 (15).

in 2 ml of nutrient broth (NB) (Oxoid, Basingstoke, England)overnight at 37°C with shaking. Aliquots (0.1 ml) of theovernight cultures were added to 2 ml of top agar containing(final plate concentrations) 3 ,M L-histidine, 3 ,uM biotin,and 500 ,AM L-arginine and were plated on plates containingVBC salts (20) and 0.4% glucose (VBC/glucose plates).Colonies were counted [by hand for plates with <100 colo-nies and with a New Brunswick (Edison, NJ) Biotran IIautomatic colony counter for plates with >100 colonies] after48 hr at 37°C. Strains carrying the pACYC184-derived plas-mids were grown in NB (Oxoid) containing chloramphenicolat 10 ,ug/ml. The 5-fluorouracil forward-mutation assayswere carried out as described for the His'-reversion assaysexcept that 5-fluorouracil and L-histidine were added to thetop agar to have final plate concentrations of 8 ,M and 100,M, respectively.The His'-reversion assay was modified slightly to test the

effect of anaerobic growth conditions. Cells were inoculatedinto 2 ml ofNB (Oxoid) containing 0.4% glucose. Half of thecultures were grown aerobically with shaking, while the othercultures were purged with nitrogen for 5 min, capped, andthen grown without shaking. All of the cultures were platedon VBC/glucose as described above. The anaerobic plateswere incubated in GasPak anaerobic containers (<0.2%oxygen according to Baltimore Biological Laboratory Micro-biology Systems specifications).

Classification of Mutations. The classification of the spon-taneous mutations was carried out as described (21). Themutants were classified as small internal deletions, intragenic

point mutants, or extragenic suppressors according to theirsensitivity to thiazole alanine. Overnight cultures grown inNB medium (Difco) were plated on VBC/glucose plates in 2ml of top agar containing biotin and L-arginine. A diskimpregnated with 20 ,ul of thiazole alanine solution (20mg/ml) was placed in the center ofeach plate. The revertantswere scored for their sensitivity to thiazole alanine by thediameter of the zone of inhibition around the disks after 12 hrat 370C.The suppressor revertants were further classified accord-

ing to their ability to plaque P22 phage carrying nonsensemutations. Overnight NB (Difco) cultures were plated on NB(Difco) plates in 2 ml of top agar. Ten-microliter drops(containing 106 plaque-forming units per ml) of three non-sense mutant phage (4262, 4266, 4491) and one wild-typephage (41) (15) were spotted onto different sections of theplate. Strains in which only mutant 4262 gave plaques wereclassified as supG, those in which only mutant 4266 gaveplaques were classified as supC/supM, and revertants inwhich all mutant phage gave plaques were classified as supB(21).

RESULTSoxyR Deletion Mutants Have High Spontaneous-Mutation

Frequencies. Several different S. typhimurium mutagenicitytester strains have been developed that measure the back-mutation frequency of specially constructed his mutants. Onetester strain (TA102) carrying the hisG428 ochre mutationwas found to be reverted by chemical oxidants, a class ofmutagens not detected by other tester strains (22). This strainhas an A-T at the mutated site in contrast to other testerstrains, which have G-C base pairs at the site of mutation (19).The oxyRI mutant allele and the oxyRA2 deletion were

transduced into a strain containing the hisG428 mutation. Thefrequency of spontaneous mutagenesis of hisG428 to His'was then tested in the two oxyR backgrounds using standardprocedures. The strain carrying the oxyRI mutant allele(TA4115) consistently showed a lower mutation frequency bya factor of 2 than the otherwise isogenic oxyR+ parent(TA4114), while the oxyRA2 deletion strain (TA4116) exhib-ited a 10-fold higher spontaneous-mutation frequency (Table2).The oxyRI and oxyRA2 alleles were also transduced into

hisG428 tester strains containing the pKM101 plasmid(TA4117-TA4119). pKM101 encodes the mucA and mucBgenes, analogs of the E. coli umuC and umuD genes, thatmake strains more susceptible to mutagenesis by chemicaloxidants, far UV light, and a number of other chemicalmutagens (22, 23). In addition, the oxyR mutations weretransduced into a uvrB deletion strain (TA4120-TA4122) aswell as into a strain carrying both the uvrB deletion and thepKM101 plasmid (TA4123-TA4125). In all cases the resultswere qualitatively similar; strains with the oxyRI allele hadlower mutation frequencies by a factor of 2, and strains withthe oxyRA2 allele had 20- to 55-fold higher spontaneousmutation frequencies compared to wild type (Table 2). Thehighest spontaneous mutation frequencies were seen withoxyRA2 strains carrying the pKM101 plasmid. An average of3102 revertants per plate arose in hisG428 oxyRA2/pKM101(TA4119) compared to 76 revertants per plate in hisG428oxyRA2 (TA4116). In oxyRA2 AuvrB strains, the presence ofpKM101 caused an increase of 5000 revertants per plate (301for TA4122 compared to 5297 for TA4125).The spontaneous-mutation frequencies in all strains were

determined also by a forward-mutation assay, which mea-sures the frequency of mutation to 5-fluorouracil resistance.This assay should provide a more general assay for all typesofmutations because resistance to 5-fluorouracil is thought toarise via loss of function mutations in the gene for uracil

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Table 2. Spontaneous-mutation frequencies in oxyRI, oxyRi,and oxyRA2 strains

His' 5-FUrrevertants mutants per

Strain per plate plate

hisG428 oxyRI 6 (42) 251 (18)hisG428 oxyRi 3 275hisG428 oxyRA2 76 1,632

hisG428 oxyR4/pKM101 57 (27) 191 (7)hisG428 oxyRl/pKM101 15 41hisG428 oxyRA2/pKM101 3,102 3,053

hisG428 AuvrB oxyR' 14 (7) 38 (6)hisG428 AuvrB oxyRI 6 15hisG428 AuvrB oxyRA2 301 4,510

hisG428 AuvrB oxyR4/pKM101 278 (10) 4,865 (10)hisG428 AuvrB oxyRl/pKM101 63 1,498hisG428 AuvrB oxyRA2/pKM101 5,297 11,684

hisG46 AuvrB oxyR4/pKM101 365 (10) 2,646 (10)hisG46 AuvrB oxyRl/pKM101 210 463hisG46 AuvrB oxyRA2/pKM101 242 5,754

Data are listed as the mean number of colonies per plate. Thenumber of experiments done for each set of strains is given inparentheses. The standard error of the mean for the following strainsis representative: hisG428 oxyR', 6 ± 4; hisG428 oxyRI, 3 ± 2;hisG428 oxyRA2, 76 ± 65; hisG428 oxyR4/pKM101, 57 ± 34;hisG428 oxyRl/pKM101, 15 ± 9; hisG428 oxyRA2/pKM101, 3102 ±1610. The number of colonies per plate varied between experiments,but each experiment was internally consistent. 5-FU', 5-fluorouracilresistance.

kinase, which could arise by deletions, insertions, or any ofthe six possible base-substitution mutations. As shown inTable 2, the effects of oxyR on spontaneous-mutation fre-quency as monitored by 5-fluorouracil resistance are quali-tatively similar to the effects observed using the His'back-mutation assay.

Finally, the spontaneous-mutation frequency was deter-mined in a strain carrying the hisG46 mutation (TA4126-TA4128). The effects of the oxyR alleles were not as pro-nounced on the His'-reversion frequency as in the hisG428background (Table 2). The hisG46 mutation arose via a G-Cto CG transversion, which can only be reverted by basesubstitutions at the COG pair and not via unlinked suppressors(24). It appears that oxyR predominantly affects the frequen-cy at which mutations arise at ANT base pairs (see below). Asexpected, the more general 5-fluorouracil resistance assayshowed a more pronounced effect of the oxyR alleles in thehisG46 background.The Spontaneous-Mutation Frequency in oxyR Deletion

Mutants Is Diminished Under Anaerobic Conditions. Reactiveoxygen intermediates generated during respiration are po-tential sources of DNA damage (2-7). Therefore, we testedthe effect of anaerobic growth on the spontaneous-mutationfrequencies (Table 3). Anaerobic incubation of the oxyRA2liquid culture and the assay plates consistently decreased thespontaneous-mutation frequency by a factor of 3, while thespontaneous-mutation frequency was decreased by a factorof 1.5 for oxyR' strains and unaffected for oxyRi strains.These results suggest that the increased spontaneous-muta-tion frequencies seen in deletions of oxyR are due, at least inpart, to the inability of the mutants to protect their DNAagainst oxidants generated during aerobic growth.We also analyzed the effects of other possible sources of

oxidation on the spontaneous-mutation frequency. Low lev-els of both hydrogen peroxide and superoxide anion are

Table 3. Spontaneous mutagenesis under anaerobic conditionsHis'-revertant colonies per

plate*hisG428/pKM101 oxyR+ oxyRl oxyRA2

Aerobic conditions 59 14 2147Anaerobic conditions 40 15 661

*Data are listed as the mean number of His+-revertant colonies perplate for eight sets of plates. The anaerobic and aerobic conditionsare described in Materials and Methods.

present in some types of bacterial growth media (25). Todetermine whether oxidants in the media might be responsi-ble for the effects of oxyR on spontaneous-mutation frequen-cy, we added 1 mg of both bovine catalase (14 units) andbovine superoxide dismutase (3000 units) per ml of the liquidculture and the assay plates. The amounts of catalase andsuperoxide dismutase used in these experiments are in vastexcess of what should be required to remove all hydrogenperoxide and superoxide that might be in the media (25).Bovine serum albumin (1 mg/ml) was included as a nonen-zymatic control. None of these treatments affected thespontaneous-mutation frequency (data not shown).The Frequency of TEA to AT Transversion Mutations Is

Increased in Deletions of oxyR. We determined the types ofmutations that are responsible for the altered spontaneous-mutation frequencies in oxyR mutant strains. In this analysiswe took advantage of simple phenotypic assays that distin-guish among the various types of revertants of the ochremutation (TAA) at hisG428. The three classes of hisG428revertants-(i) base-substitution mutations at the hisG428site, (ii) small in-frame deletions at the hisG428 site, and (iii)base-substitution mutations at extragenic tRNA suppressorloci-can be distinguished on the basis of their sensitivity tothe histidine analog thiazole alanine (26). The tRNA suppres-sor mutations can be further classified as supG (UUU toAUU), supC/supM (AUG to AUU), or supB (GUU to AUU)based on the ability ofthe tRNA suppressor strains to supportthe growth of a diagnostic set of P22 phage that carrynonsense mutations blocking phage growth (15, 21).

Table 4. Classification of His' revertants

Type of mutation oxyR+ oxyRi oxyRA2hisG428

Total number analyzed 123 125 119Extragenic suppressorssupG (TSAR-* AT) 1 0 40 (401)supC/M(CGA-*AT) 1 0 9 (91)supB (C-G-.TA) 0 0 0Uncharacterized 0 0 6

Intragenic point mutants(TMA - ?) 2 1 (24,) 16 (8t)

Small internal deletions 1 2 (2 T) 5 (5 T)

hisG428/pKM101Total number analyzed 112 108 114Extragenic suppressorssupG (T-A-AT) 8 3 (34 ) 1170 (146 )supC/M (C-G. AT) 20 2 (10 1) 572 (291)supB (C-G -* T-A) 0 1 27Uncharacterized 3 1 109

Intragenic point mutants(T-A ?) 24 5 (5, ) 1197 (501)

Small internal deletions 2 3 (2 T) 27 (14 T)The data are expressed as the average number of each type of

revertant per plate. The factor increase (T) or decrease ( I)compared to oxyR' is given in parentheses.

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The analysis (Table 4) showed that there was an increasein the frequency of at least three of the six possible base-substitution mutations (TEA to ANT transversions, COG to A-Ttransversions, and C&G to TEA transitions) and in the frequen-cy of small deletions in hisG428 oxyRA2 (TA4116) strainscompared to the hisG428 oxyR' strain. However, the largestincrease was in the frequency of TEA to ANT transversions,which give rise to supG suppressors in our assay (up 40-fold).TEA to ANT transversions are the type of mutation mostfrequently caused by oxidative mutagens (21). The constitu-tive oxyRi mutant showed a 2-fold increase in the frequencyof deletion revertants, while the number of revertants in allother classes was decreased. The mutation spectrum was alsotested in the hisG428/pKM101 background and found to besimilar to the spectrum in the hisG428 background (Table 4).Again hisG428 oxyRA2/pKM101 showed an increase in alltypes of revertants compared to hisG428 oxyR+/pKM101.The number of TEA to ANT transversions was increased themost (146-fold).

Reintroduction of the oxyR Gene into oxyR Deletion MutantsDiminishes the Mutation Frequency. Introduction ofa plasmidcarrying the E. coli oxyR gene on a 1.5-kb EcoRV-BamHIfragment into oxyRA2 strains (TA4112 and TA4119) restoredthe spontaneous-mutation frequency to near wild-type levels,while the plasmid vector alone had little effect on themutation frequency in both strains (Table 5). This demon-strates that the increased spontaneous-mutation frequency inthe oxyR deletion strains is due to the absence of the oxyRgene and is not due to the deletion of neighboring genes.

Overexpression of Some Genes Controlled by oxyR Decreas-es the Spontaneous-Mutation Frequency in the oxyR DeletionMutant. To determine whether the effect ofoxyR deletions onspontaneous mutagenesis was a result of the uninducibility ofoxyR-regulated genes, we tested whether the high spontane-ous-mutation frequency in oxyR deletion strains could bedecreased by the introduction of multicopy plasmids carryingthe oxyR-regulated katG, ahp, or sodA genes. Introduction ofeither katG or ahp on multicopy plasmids diminished thefrequency to near wild-type levels (Table 5). Introduction ofthe sodA gene diminished the mutation frequency by a factorof 5 in hisG428 oxyRA2/pKM101 (TA4119) but slightly

Table 5. Overexpression of some genes controlled by oxyRdecreases the spontaneous-mutation frequency in oxyRA2

His+ revertantsStrain per plate

hisG428 oxyR' 6hisG428 oxyRi 3hisG428 oxyRA2 76hisG428 oxyRA2/pACYC184 (vector) 38hisG428 oxyRA&2/pAQ5 (oxyR) 4hisG428 oxyRA2/pAQ6 (sodA) 90*hisG428 oxyRA2/pAQ7 (katG) 9hisG428 oxyRA2/pAQ8 (ahp) 6hisG428 oxyR+/pKM101 57hisG428 oxyRI/pKM101 15hisG428 oxyRA2/pKM101 3102hisG428 oxyRA2/pKM101/pACYC184 (vector) 946hisG428 oxyRA2/pKM1J1/pAQS(oxyR) 33hisG428 oxyRA2/pKM101/pAQ6 (sodA) 196*hisG428 oxyRA2/pKM101/pAQ7 (katG) 47hisG428 oxyRA2/pKM101/pAQ8 (ahp) 31

Data are given as the mean number of His' revertants per plate for>10 sets of plates. The construction of the plasmids is described inMaterials and Methods.*Manganese sulfate (100 /iM) was added to the liquid cultures andplates to allow full overproduction of the Mn-containing superoxidedismutase encoded by sodA. The added manganese sulfate alone didnot affect the spontaneous-mutation frequency in any other strain.

increased the mutation frequency in hisG428 oxyRA2 (TA-4116). The levels of superoxide dismutase were found to beincreased 10- to 20-fold (depending on the strain) as seen byactivity-stained gels. The level of catalase activity was alsofound to be increased 10- to 20-fold, and the level of alkylhydroperoxide reductase activity was increased 4-fold.We also determined the spontaneous-mutation frequency

in strains carrying null mutations in two genes regulated byoxyR (ahp and katG). A deletion ofkatG (14), which codes forcatalase, and a TnJO insertion in ahp, the locus encoding apreviously unreported alkyl hydroperoxide reductase (F.Jacobson, G.S., L. Tartaglia, and B.N.A., unpublishedresults), were transduced into tester-strain backgrounds(TA4188 and TA4189). Neither mutation altered the rate ofspontaneous mutagenesis compared to wild type (data notshown).

DISCUSSIONWe have shown that strains of S. typhimurium compromisedin their ability to induce a defense response against oxidativestress because of a deletion of the oxyR locus have highspontaneous-mutation frequencies. The spontaneous-muta-tion frequencies are increased most in oxyRA2 strains car-rying pKM101-encoded mucA and mucB and in oxyRA2strains carrying pKM101 together with deletions ofuvrB. Thehigh spontaneous-mutation frequencies are partially oxygen-dependent, indicating that the additional mutations occurringin oxyRA2 strains are probably due to oxidative DNAdamage. This conclusion is further supported by the obser-vation that the type ofmutation whose frequency is increasedthe most in oxyR deletion strains is a T-A toAT transversion,the base substitution most frequently caused by oxidativemutagens. Our results suggest that for every 6 visible spon-taneous-mutation events occurring in hisG428 oxyR+, thereare about 5300 spontaneous DNA-damaging events that aredue to oxidative damage and normally are prevented by thepresence of the oxyR system. Presumably these events arelethal unless they are converted to mutations by mucA ormucB or are repaired by uvrB.The large effect of the pKM101 plasmid on the spontane-

ous-mutation frequency in oxyR deletion strains and theincreased rate of T-A to AT transversions suggests that theincreased mutation frequency in oxyRA2 is the result ofSOS-dependent replication past apurinic or apyrimidinic(AP) sites. Numerous in vitro and in vivo studies have shownthat AP sites are intermediates in mutagenesis (reviewed inref. 27). In E. coli, replication past AP sites requires SOSinduction and umuC, recA, and recF (28, 29). The DNApolymerase preferentially inserts dAMP and secondarilydTMP opposite the AP sites (27, 30). Therefore, we proposethat active oxygen species in the cell are forming damagedbases, many of which are converted to AP sites. In theabsence of uvrB repair, DNA polymerase replicating past theAP sites preferentially incorporates dAMP ordTMP oppositethe damaged sites, causing some mutagenic changes. Forexample, formamidopyrimidine, a result of oxidative damageto adenosine (31), may be removed to generate an AP site(32). If dAMP were incorporated opposite the AP site duringreplication, this modification would generate a T-A to ATtransversion.Our results indicate that the protective effect ofoxyR is due

to the induction of enzymes capable of scavenging activeoxygen species. These enzymes must act synergistically tolower the intracellular pool of active oxygen species, sincethe high mutation frequency in oxyR deletion strains waslowered by overproducing oxyR-induced catalase, alkylhydroperoxide reductase, or superoxide dismutase, thoughsingle mutations in the genes encoding catalase and alkylhydroperoxide reductase (katG and ahp) had little effect on

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the spontaneous-mutation frequency. We expect strains withmultiple mutations in the oxyR-regulated genes will haveincreased spontaneous-mutation frequencies. It is also likelythat deletions of the sodA gene encoding Mn-containingsuperoxide dismutase, an activity found to be increased2-fold in oxyRI mutants, will increase the spontaneous-mutation frequency. A sodA mutant in E. coli shows a 9-foldincrease in spontaneous-mutation frequency (9).The oxyR gene product may also act to induce DNA repair

enzymes that specifically act on oxidatively damaged DNA.Exonuclease III and endonuclease IV can remove 3' blocksfrom DNA damaged by hydrogen peroxide (33). Chan andWeiss have recently found that the expression of the geneencoding endonuclease IV is induced by various oxidants(34). Possibly further characterization of oxyR-regulatedgenes will reveal oxyR control of such DNA repair enzymes,which may help prevent spontaneous mutagenesis occurringas a result of oxidative DNA damage.

We thank Daniele Touati for the sodA clone and Peter Loewen forthe katG clone. This work was supported by Grant GM19993 fromthe National Institutes of Health and National Cancer InstituteOutstanding Investigator Grant CA39910 to B.N.A., by Center GrantES01896 from the National Institute of Environmental Health Sci-ences, and by a grant from the National Foundation for CancerResearch (Bethesda, MD) to H.S.; G.S. and M.F.C. were supportedby Training Grant GM07232 from the National Institutes of Health.

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