Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Mutation Research 435 Ž1999. 245–254 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres

Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid Karen A. Schouten, Bernard Weiss

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Department of Pathology and Laboratory Medicine, Emory UniÕersity School of Medicine, Atlanta, GA 30322 USA Received 3 June 1999; accepted 27 July 1999

Abstract Endonuclease V Ždeoxyinosine 3X-endonuclease. of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA’s containing hypoxanthine, uracil, or mismatched bases. An endonuclease V Ž nfi . mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfiq strains, they displayed a high frequency of A:T G:C, and G:C A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N 6-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T G:C mutations in an nfi mutant, even though AlkA Ž3-methyladenine-DNA glycosylase II. has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C A:T transitions. These mutations could not be explained by cytosine deamination because an ung Žuracil-DNA N-glycosylase. mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination. q 1999 Elsevier Science B.V. All rights reserved.

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Keywords: Endonuclease V; nfi gene; Nitrous acid; Deoxyinosine 3 -endonuclease; Nitrosative deamination

1. Introduction

AbbreÕiations: Endo V, endonuclease V; PCR, polymerase chain reaction ) Corresponding author. Glenn Memorial Building, 69 Butler St., S.E., Atlanta, GA 30303, USA. Tel.: q1-404-616-0602; fax: q1-404-616-7455; e-mail: [email protected].

Endonuclease V ŽEndo V. of Escherichia coli w1,2x, or deoxyinosine 3X-endonuclease w3x, is a putative DNA repair enzyme encoded by the nfi gene w4,5x. It cleaves the second phosphodiester bond 3X to a deoxyinosine Ži.e., deaminated deoxyadenosine. in DNA. The enzyme was originally described as being

0921-8777r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 4 9 - X

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K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

specific for single-stranded DNA, uracil-containing DNA, and DNA’s that were treated with acid, alkali, OsO4 , UV radiation, or 7-bromomethylbenzŽ a.anthracene w1,2x. Subsequently, it was found to cleave DNA near deoxyinosine Žhypoxanthine deoxyribonucleoside., abasic sites, urea glycoside residues, sin gle-base mismatches, loops, pseudo-Y structures, hairpins, and flaps w3,5,6x. Despite this broad specificity, it is a simple protein of relatively low molecu lar weight Ž Mr s 24,672., suggesting that it may be primitive and therefore widespread. The properties of the purified enzyme indicated that it might have a role in repairing deaminated bases in DNA, which are a cause of both spontaneous and chemically induced mutations. Nitrous acid ŽHNO 2 . is a common environmental and metabolic mutagen. It can attack polynucleotides to deaminate guanine to xanthine, adenine to hypoxanthine Žthe base in deoxyinosine., and cytosine to uracil w7x. Endo V can recognize the resulting mismatches in DNA w6x. It also specifically recognizes deoxyinosine w3x even if the nucleoside should become correctly paired with deoxycytidine after a round of replication. However, an nfi mutant is not unusually sensitive to killing by HNO 2 ; instead it is slightly more resistant w8x. This paradoxical result suggested that Endo V may actually contribute slightly to the lethality of HNO 2 , probably by causing breakage of DNA at lesions, and that HNO 2 does not kill cells primarily through Endo V-remediable lesions. An nfi mutant displayed a high frequency of HNO 2-induced mutations to Str r w8x. Previously, mutations to Str r were found to be transitions or transversions at A:T base pairs within the rpsL gene w9x. This specific mutator effect is consistent with the hypothesis that an nfi mutant is defective in the removal of DNA hypoxanthine, which then pairs with cytosine during replication to produce A:T G:C transition mutations. On the other hand, HNO 2induced mutations to valine or azauracil resistance, which can arise by many different base changes, were only mildly elevated in an nfi mutant. These results suggested that Endo V helps to repair specific base alterations caused by HNO 2 . In this study, we explore the specificity of spontaneous and HNO 2-induced mutagenesis in an nfi mutant. We employ a set of mutation indicator strains

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having trp and lac mutations that revert to wild type through known base-pair changes.

2. Materials and methods 2.1. Bacterial strains The bacterial strains used were derivatives of E. coli K-12 ly. The trp mutants were those previously described w10,11x. They were obtained from E.C. Cox and R.G. Fowler and had the following additional genotype: Fy leu argE5 his thr rpsE. The lacZ mutant strains, provided by C.G. Cupples w12,13x, had the following genotype: ara DŽ proBlac . X l l l ŽFX lacI378 lacZ proBq .. Derivatives of these strains were prepared by generalized transduction using P1 dam reÕ6 w14x. Strain BW1185 w8x was used as the donor for nfi-1
K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

were treated for 6 min at 378C with 40 mM NaNO 2 in 0.1 M sodium acetate buffer ŽpH 4.6., as previously described w8x. nfiq and nfi-1
3. Results 3.1. Construction of nfi mutant deriÕatiÕes of mutation tester strains The parental nfiq strains used in this study were trp and lacZ mutants that are often used for studies

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of mutagenic specificity in E. coli because they revert by known base-pair changes ŽTable 1.. The nfi-1
Table 1 Frequencies of spontaneous mutants in nfiq and nfi-1
trpA11 trpA58 trpA58 alkA lacZŽCC106. trpA223 trpA446 lacZŽCC102. lacZŽCC102. ung trpA3 lacZŽCC105. trpA78 lacZŽCC101. lacZŽCC104. lacZŽCC103. lacZŽCC107. lacZŽCC108. lacZŽCC109. lacZŽCC110. lacZŽCC111. trpA21 trpA9813 trpA540 trpE9777

Pathway to full full revesiona

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nfiq strain

A:T G:C c A:T G:C A:T G:C A:T G:C 6-MeA:T G:C or C:G G:C A:T G:C A:T G:C A:T A:T T:A A:T T:A A:T C:G A:T C:G G:C T:A G:C C:G q1G Fs y1G Fs y2Ž –C–G– . Fs q1A Fs y1A Fs q1 Fs q1 Fs q1 Žorq2?. Fs y1 Fs

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Number of revertantsb

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nfi-1 strain

0 1 1 1 4

1 2 1 0 4

1 23 370 2 11 2 4 36 0 702 131 817 48 347 6 0 9 35

2 23 281 2 1 4 3 94 0 588 178 1031 56 367 5 1 25 37

a References: lacZ mutation of strain CC104 w12x, trp frameshift mutations w22x, trpA11 and trpA223 Žthis study and Ref. w11x. other trp alleles w11x. b Median values for either 9 or 18 cultures containing 3=10 9 cells. c The trpA11 allele was previously described as a probable C:G G:C indicator Žsee text..

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K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

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the nfi-1
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The spontaneous deamination of cytosine in DNA produces G:U mismatches that result in G:C A:T transitions if they are not repaired. Most of this repair is initiated by uracil-DNA N-glycosylase w23x, the product of the ung gene. Because G:U mismatches are also recognized by Endo V, we examined the relative roles of the two enzymes. Among the strains tested was an ung mutant containing lacZŽCC102., which reverts to the wild type by a G:C A:T transition. As expected from previous results w18x, the ung mutation produced a marked increase in the frequency of spontaneous G:C A:T transitions in the nfiq and nfi mutant strains Ž12-fold and 16-fold, respectively.. In contrast, the introduction of an nfi mutation into either the ungq or ung mutant strain produced no increase in mutation frequency. Therefore, unlike uracil-DNA glycosylase ŽUng., Endo V does not appear to play a significant role in the repair of G:U base pairs in the DNA of untreated cells.

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3.4. Nitrous acid-induced mutations The mutation indicator strains were then treated with HNO 2 . Under the experimental conditions, the survival of the trp mutants ranged from 3% to 21%, and that for the lacZ indicator strains was 7% to 26%. Consistent with previous results w8x, the survival of each nfi mutant was either about equal to that of its nfiq parent or up to several times greater than it. The base-pair changes listed in Tables 1 and 2 are those known to result in full reversion. For the trp alleles, a full revertant was operationally defined w24x as a Trpq derivative that is relatively resistant to 5-methyltryptophan, that produces colonies as large as the wild type after 48 h of growth, and that does not accumulate indoleglycerol in the medium. HNO 2-induced Trpq revertants were tested to confirm that they were not contaminants or pseudorevertants. Twenty-four colonies from each tested strain Žfour colonies arising from each of six treated samples. were repurified by streaking on tryptophan-deficient media. All of them retained the parental arg and thr genotypes and failed to accumulate indoleglycerol. For the lac alleles, partial reversion was not a problem w12,13x. The results of HNO 2-induced mutagenesis are shown in Table 2. Note that in this table, the values for the untreated cultures represent the background frequencies for individual samples that were to be treated with HNO 2 . Each of these values is for a single culture; therefore they are not as representative of the spontaneous mutation frequencies as the values in Table 1. The data sets for each of the treated strains were not suitable for statistical analysis because they did not follow any recognizable distribution pattern. Therefore, values for the untreated samples are also given to assist in the interpretation of the results. The net results, i.e., HNO 2induced mutations in nfiq and nfi mutant strains, are presented in the last two columns of Table 2. To interpret this data, we must ask the following question: For any indicator allele, are the differences in induced mutations large with respect to the background frequencies in the untreated cultures? The results ŽTable 2. indicated that the nfi mutation greatly enhances the frequency of two types of HNO 2-induced base-pair changes: A:T G:C transitions Ž trpA11, trpA58, and lacZŽCC106.. and G:C

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Table 2 The effect of an nfi mutation on the frequency and specificity of HNO 2 -induced mutations Abbreviations: 6-MeA, 6-methyladenine; Fs, frameshift. Relevant genotype

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nfi

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A:T G:C A:T G:C A:T G:C A:T G:C 6-MeA:T G:C or C:G G:C A:T G:C A:T G:C A:T A:T T:A A:T T:A A:T C:G A:T C:G G:C T:A G:C C:G q1G Fs y1G Fs y2Ž –C–G– . Fs q1A Fs y1A Fs q1 Fs q1 Fs q1 Žorq2?. Fs y1 Fs

strain

nfi-1 strain

Untreated

qHNO 2a

Untreated

qHNO 2

4 0 6 0 5 0 20 375 0 6 0 6 46 0 185 56 2013 95 720 67 3 11 358

11 1 7 2 6 1 140 580 2 25 58 111 57 78 441 1037 1355 72 920 18 26 30 609

2 2 10 0 2 5 12 144 0 8 3 20 217 0 180 21 501 23 1315 55 5 20 80

251 535 188 34 7 520 2790 7800 5 22 111 95 191 21 470 647 518 11 1025 33 36 84 298

nfiq strain

nfi-1 strain

7 1 1 2 1 1 120 205 2 19 58 105 11 78 256 981 0 0 200 0 23 19 251

249 533 178 34 5 515 2778 7656 5 14 108 75 0 21 290 626 17 0 0 0 31 64 218

K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

trpA11 trpA58 trpA58 alkA lacZŽCC106. trpA223 trpA446 lacZŽCC102. lacZŽCC102. ung trpA3 lacZŽCC105. trpA78 lacZŽCC101. lacZ ŽCC104. lacZŽCC103. lacZŽCC107. lacZŽCC108. lacZŽCC109. lacZŽCC110. lacZŽCC111. trpA21 trpA9813 trpA540 trpE9777

HNO 2-induced revertants Žno.rml. b

Revertants Žno.rml.

a qHNO 2 , treated with 40 mM NaNO 2 at pH 4.6 for 6 min; median values for six treated samples that were grown to saturation Žsee Section 2.. Untreated samples were incubated in the buffer alone Žat pH 4.6. without significant loss of viability w8x. b The frequency of HNO 2 -induced revertants is the difference between the values obtained for the HNO 2 -treated sample and the corresponding untreated sample.

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K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

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A:T transitions Ž trpA446 and lacZŽCC102... The nfi mutation had no major effect on transversions. Among the tester strains were frameshift indicators. Frameshift mutations usually occur in sequences of repeated nucleotides and may be generated by slippage of the template strand with respect to the growing daughter strand w25x. This process should create a substrate for Endo V, which cleaves at looped-out nucleotides w5x. However, the nfi mutation did not promote a large number of frameshift mutations either in untreated cells ŽTable 1. or in ones treated with HNO 2 ŽTable 2.. 3.5. Nature of the trpA11 and trpA223 mutations Based on their previous characterizations, the trpA11 and trpA223 alleles did not give results that were consistent with those obtained with similar indicators. The trpA11 allele was presumed to be an indicator of G:C C:G transversions; it has been used for that purpose Že.g., see Ref. w10x. even though that assignment had only been only tentative w26x. However, whereas the trpA11 allele demonstrated a high frequency of HNO 2-induced reversion in an nfi mutant background, the lacZŽCC103. allele, a known G:C C:G indicator, did not. Similarly, the trpA223 allele, originally described as an A:T G:C indicator, failed to show the high rate of HNO 2-induced reversions that was seen for other A:T G:C indicators in an nfi mutant host ŽTable 2.. The base-pair changes producing reversion of these trp alleles had been merely inferred from protein sequences years before the availability of DNA sequencing. Therefore, we examined the DNA sequences. The trpA gene of a trpA11 mutant was amplified by PCR, and its sequence was determined together with that of a full revertant. The trpA11 mutation, in codon 49 of the ORF Ždescribed originally as codon 48 of the polypeptide. had been tentatively designated as GAG ŽGlu. CAG ŽGln. w26x, which would require a G:C C:G base-pair change for reversion. We found that whereas the sequence from a full revertant was indeed GAG, matching that of the wild type gene ŽGenBank AE000224., the mutant codon was actually AAG ŽLys.. Therefore, trpA11 is an A:T G:C indicator Žas designated in Tables 1 and 2.. Its high rate of HNO 2-induced reversion is con-

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sistent with the results obtained with the other A:T G:C indicators ŽTable 2.. Full phenotypic reversions of trpA223 were originally described as occurring either by A:T G:C or A:T C:G w24x. However, an examination of the sequence of the wild type gene ŽGenBank AE000224. revealed that the mutant A:T base pair is within GATC, the dam methylation sequence w27x. Therefore, it contains N 6-methyladenine rather than adenine, and as indicated in Table 1 and 2, trpA223 must revert by changes in a 6-MeA:T rather than an A:T base pair. In an nfi mutant, the relative resistance of trpA223 to HNO 2-induced reversion is consistent with the known resistance of N 6-methyladenine to nitrosative deamination w28x Žsee Section 4..

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3.6. RelatiÕe effects of nfi, alkA, and ung mutations AlkA Ž3-methyladenine-DNA glycosylase II. has hypoxanthine DNA-glycosylase activity w29x. Therefore, we compared nfi and alkA mutants. trpA58 reverts by A:T G:C base-pair changes, which would be expected to occur from unrepaired deaminated adenine Ži.e., hypoxanthine. in DNA. Under our experimental conditions, the alkA mutation did not significantly increase the frequency of either spontaneous ŽTable 1. or HNO 2-induced ŽTable 2. reversion of trpA58, thus confirming a previous report of work with a different A:T G:C indicator allele w30x. Under the same conditions, however, the nfi mutant demonstrated over a 200-fold mutagenesis by HNO 2 treatment, which was not further increased by an alkA mutation ŽTable 2.. Therefore, AlkA appears to have no demonstrable role in the repair of these Endo V-remediable lesions. An ung mutant was similarly tested. Because of a defective repair of deaminated cytosine Ži.e., uracil. in DNA, it produced a high incidence of spontaneous G:C A:T reversions of lacZŽCC102. ŽTables 1 and 2. as expected w18x. On the other hand, an nfi mutation produced no such increase. Therefore, in vivo, Ung is much more active than Endo V in the repair of deaminated cytosine. HNO 2-induced G:C A:T reversions of lacZŽCC102. were then examined ŽTable 2.. An ung mutation resulted in only a small increase in these mutations in both the nfiq and nfi mutant derivatives Žabout twofold and three-

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K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

fold, respectively; Table 2.. However, an nfi mutation produced a much greater increase in these reversions in both ungq and ung mutant derivatives Ž22-fold and 36-fold, respectively; Table 2.. Thus, most of the HNO 2-induced G:C A:T mutations that can be prevented by Endo V cannot be prevented by Ung, despite the greater cellular activity of the latter enzyme against deaminated cytosine in DNA. Therefore, most of these mutations are probably not caused by deaminated cytosine. Endo V must recognize another lesion caused by HNO 2 at these G:C base pairs Žsee Section 4..

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4. Discussion 4.1. Mutations produced by HNO2 The results of this study indicate that Endo V plays an important role in protecting E. coli from the mutagenic effects of HNO 2 . The major source of HNO 2 and nitrite in nature is probably from the bacterial reduction of nitrate fertilizers; nitrate is the preferred electron acceptor during the anaerobic growth of many bacteria. The chief nitrosating agent derived from HNO 2 is its anhydride, N2 O 3 w31x. Because N2 O 3 forms from the undissociated acid, mutagenesis by HNO 2 is most efficient at a low pH, such as that used as in this study. The nitrate reductases of E. coli and related bacteria catalyze reactions leading to nitrosation, which may be mutagenic in repair-deficient cells w32x. The N-nitroso compounds that are mutagenic by-products of anaerobic metabolism may be thought of as the counterparts of the mutagenic reactive oxygen species that arise during aerobic metabolism. The two major DNA lesions produced by HNO 2 Žreviewed in Ref. w33x. are deaminated bases and alkylated bases. In addition, HNO 2 produces G–G crosslinks, protein–DNA crosslinks, and, through an unknown mechanism, T C mutations. The oxidative deamination of DNA bases occurs through the formation of unstable nitroso derivatives of the exocyclic amines. Alkylation is mediated by the nitrosation of cellular secondary amines and secondary amides, which thereby become alkylating agents. These alkylating agents may form even under

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physiological conditions and may thus be a major cause of endogenous DNA damage and an important reason for the evolution of methytransferases involved in DNA repair w32x. 4.2. Analysis of spontaneous mutations In a previous study w8x, an nfi mutant was found to have about twofold elevated frequencies for several types of forward mutation. Our hypothesis was that Endo V might recognize only specific lesions, such as deaminated adenine, that produce only a fraction of spontaneous mutations. If so, then a mutator effect of nfi would be magnified by systems that detect specific base-pair changes, such as the ones used in this study. However, we did not observe any marked effect of nfi on spontaneous mutagenesis ŽTable 1.. Therefore, at least under our experimental conditions, i.e., aerobic growth in a rich medium, nfi mutants do not display a strong mutator phenotype. When presented with a double-stranded oligonucleotide substrate containing mispaired or unpaired bases, purified Endo V specifically cleaves that strand in which the lesion is closest to the 5X end w6x. In the cell, free 5X ends are to be found in Okazaki fragments, which are formed during synthesis of the lagging strand. Therefore, it was postulated that Endo V might function to correct replication errors, such as mismatches and frameshifts, during lagging-strand DNA synthesis w5x, and this was a major reason for the inclusion of frameshift indicators in our study. However, even if other error-correction pathways were inoperative, we should expect Endo V to correct no more than half the errors, i.e., only those on the lagging strand, thereby producing no more than twofold differences in the frequency of some types of spontaneous mutations. Such small differences could not be reliably detected in this study in which the low values and irregular distribution of the results precluded statistical analysis. The most sensitive way to detect these differences might be with a system that discerns strand-specific bias for mutations in strains deficient in methyl-directed mismatch repair w34x. Thus, although our spontaneous mutation data indicate that there is no strong mutator phenotype for nfi, they do not rule out a

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possible role in the repair of as much as 50% of some types of spontaneous lesions. 4.3. A:T

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The finding that is most easily explained is the high rate of HNO 2-induced A:T G:C transitions: HNO 2 deaminates deoxyadenosine to deoxyinosine Žhypoxanthine deoxyribonucleoside., and Endo V is a deoxyinosine 3X-endonuclease. In the nfi mutant, the unremoved hypoxanthine would pair preferentially with cytosine during the next round of DNA replication, and ultimately, G:C would replace the original A:T. Therefore, the results are consistent with the hypothesis that Endo V helps to rid the HNO 2-treated chromosome of mutagenic deaminated adenine. When mutagenic specificity is found in vivo, we are usually not certain which of the two bases in a pair is altered by the mutagenic agent. T C transitions have been observed in HNO 2-treated singlestranded phage DNA w35x. Thus, it is possible that T, rather than A, is damaged in an unknown manner to produce A:T G:C mutations. This uncertainty was resolved by our results with the trpA223 nfi mutant. Unlike other A:T G:C indicator strains, it did not display a high frequency of HNO 2-induced reversion. It differs from those strains in that the adenine at the mutation site is methylated. The exocyclic group of this adenine is therefore a secondary amine rather than a primary one, and it is likely to form a relatively stable nitroso derivative rather than to be deaminated by HNO 2 w31x; methyladenine has, in fact, been demonstrated to be relatively resistant to nitrosative deamination w28x. This result indicates that A rather than T is the source of the Endo V-remediable lesion that produces A:T G:C transitions after HNO 2 mutagenesis. Base-excision pathways provide possible alternatives for the removal of deaminated adenine from DNA. Three hypoxanthine-DNA glycosylases have been reported in E. coli w29,36,37x, but it has not been established unequivocally whether or not some may be the same. One such glycosylase is a minor activity of AlkA Ž3-methyladenine-DNA glycosylase II.. Although crude extracts of alkA mutants lacked detectable hypoxanthine-DNA glycosylase w29x activ-

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ity, it is normally at such a low level w36x, that it may be difficult to detect a second enzyme. An alkA mutant was reported to have no increase in mutagenesis by HNO 2 w30x. We have obtained similar results and demonstrated in addition that an alkA nfi mutant is no more defective than an nfi mutant. Therefore, nfi, but not alkA, appears to be indispensable for protection against HNO 2-induced A:T G:C mutations.

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4.4. G:C

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An nfi mutation also stimulated HNO 2-induced G:C A:T transitions. At first, it would seem easy to attribute this result to the ability of HNO 2 to deaminate cytosine. Endo V can recognize G:U and cleave near the uracil in a relatively strand-specific manner w6x. It is therefore a candidate for the first step of a nucleotide excision pathway for this mutation. However, in vitro, this activity is only a fraction of that on deoxyinosine-containing substrates. We found that Ung is relatively more important than Endo V in preventing spontaneous G:C A:T transitions, whereas the opposite is true for HNO 2-induced mutations. Thus, the two enzymes must repair different lesions that can produce G:C A:T transitions. Ung recognizes uracil; therefore, Endo V must recognize something else. One possibility is that Endo V might work on alkylated bases. It has been suggested that HNO 2 might nitrosate an endogenous alkylamine or secondary amide to generate an alkylating agent that would produce O 6-alkylguanine in DNA, thereby causing G:C A:T transitions. However, results consistent with this effect have only been obtained in methyltransferase-deficient mutants or when methylamine or methylurea was added to the medium together with nitrite w32x. In addition, Endo V was only 40% more active on DNA that had been alkylated with methyl methanesulfonate than on untreated DNA w2x. Deaminated guanine, i.e., xanthine, remains the most likely possibility for the Endo V-remediable lesion leading to G:C A:T transitions after HNO 2 treatment. Guanine is deaminated by HNO 2 faster than the other bases in DNA; the relative rates are about 4:2:1 for G, C, and A, respectively w7x. Xanthine is ionized at neutral pH and therefore pairs

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K.A. Schouten, B. Weiss r Mutation Research 435 (1999) 245–254

poorly, hindering DNA replication w38–40x; therefore, the cell must be able to deal with xanthine as well as with hypoxanthine in DNA. The failure of deoxynebularine to be recognized by Endo V suggested that the 6-keto group of the purine is important for enzyme recognition w6x. This feature is common to both xanthine and hypoxanthine. It is therefore reasonable to suspect that Endo V might protect DNA against mutagenesis by the HNO 2-induced deamination of guanine as well as that of adenine. Studies with homoribopolymers indicated that X pairs with A and with U but not with C w38x. Therefore, the deamination of guanine should be invariably mutagenic. Guanine is, indeed, a mutagenic target of HNO 2 : HNO 2 produces G A transitions in single-stranded phage DNA w35x. The incorporation of T opposite X could explain this result as well as our observed G:C A:T transitions. In vitro, some DNA polymerases allow this mispairing w39,40x after overcoming an initial replication block. However, in vivo, the specific pairing of xanthine during DNA replication would depend on the selectivity of the replisome, and this has not been determined for E. coli. It should be emphasized, that whatever is the mutagenic lesion at the G:C site, it must be recognized by Endo V in a strand-specific manner. If either strand were cleaved with equal probability, then only 50% of the lesions would be removed before the first replication. Thus, nfi could have no more than a twofold mutator effect. However, we observed a 200- to 500-fold mutagenic enhancement in HNO 2-treated cells. Therefore, Endo V must specifically cleave the DNA strand that contains the altered base in the G:C pair, just as it specifically cleaves a hypoxanthine-containing strand. Prompted by these results Y.W. Kow Žpersonal communication. found that purified Endo V does cleave near xanthine residues in oligodeoxyribonucleotide duplexes. The overall results indicate a role for Endo V in helping to protect the cell against some of the mutagenic effects induced by HNO 2 , in particular the deamination of adenine and probably of guanine. It will be of interest to see if some of these effects can be reproduced under more physiological conditions, such as during anaerobic growth with nitrate or nitrite as an electron acceptor.

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