Proc. Nati Acad. Sci. USA Vol. 79, pp. 4133-4137, July 1982 Genetics

Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation or N-acetoxy-N-2-acetylaminofluorene (excision repair/postreplication repair/plasmid pKO482) S. E. SCHMID, M. P. DAUNE, AND R. P. P. FUCHS* Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Laboratoire de Biophysique, 67084 Strasbourg Cedex, France Communicated by James A. Miller, March 29, 1982 ABSTRACT Plasmid DNA was modified in vitro to various The plasmid used in this system, pKO482, contains a gene extents with N-acetoxy-N-2-acetylaminofluorene or UV irradia- coding for 3lactamase, which provides for ampicillin resis- tion. The modified plasmid DNAs were then used to transform tance, and a second gene, galK, which codes for the enzyme Escherichia coli strains having different repair capabilities. Both galactokinase. All of the recipient bacterial strains used were survival and mutagenesis frequencies of the plasmid were mea- ampicillin sensitive and unable to metabolize galactose due to sured as a function ofthe number oflesions per plasmid molecule. a galK mutation in their own genomic DNA. Transformed cells The majority ofN-2-acetylaminofluorene (AAF) adducts, like thy- were selected on MacConkey's agar containing ampicillin and mine dimers, were repaired by the excision (uvrA+-dependent) galactose. Complementation of the galK mutation of the host pathway. In rec' strains, dose-dependent mutagenesis occurred cell the of the allows in either AAF- or UV-modified plasmid DNA. This is in contrast by galactokinase gene plasmid galactose with results obtained in recA- strains, in which only AAF adducts fermentation and the formation ofa red colony on MacConkey's gave rise to a lower, but dose-dependent, mutagenesis frequency. agar. Mutation in the plasmid galK gene results in a visually In these recA- strains there was no UV mutagenesis. Unlike what distinguishable white colony. is observed with phages, induction of the "SOS" functions by UV We studied both plasmid survival and mutation frequency irradiation ofthe bacteria prior to transformation did not increase of the galK gene in pKO482 modified by either UV irradiation the survival or the mutagenesis of the plasmid. or N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF). Results obtained by using host cells with wild-type DNA repair capac- Two major types of DNA repair that have been found in Esch- ities are compared to those obtained by using uvrA, recA, and erichia coli are excision repair and postreplication repair (refer uvrA recA mutant strains. In addition, the effect of UV induc- to refs. 1-3 for excellent reviews). Excision repair is dependent tion of SOS functions was also studied. upon the product of the uvrA' gene along with several other gene products and is generally considered accurate and not in- MATERIALS AND METHODS volved in the formation of mutations. Postreplication repair is Bacteria and Plasmid DNA. The E. coli strains used were dependent on the recA+ gene product. This gene product ap- AB 1157 (wild-type strain), AB 1886 (uvrA-), AB 2463 (recA-), pears to be involved in the control ofexpression ofother genes and AB 2480 (uvrA- recA-) (6). All four strains carry a mutation coding for two interrelated processes that can operate to repair in the galK gene. Plasmid pKO482, 2.8 X 106 daltons, was con- gaps left in daughter strand DNA molecules after the replication structed (7) and generously supplied by K. McKenney, H. Shi- of DNA that contains damage left unrepaired by excision repair. matake, and M. Rosenberg (National Cancer Institute, Be- These two interrelated postreplication processes are recom- thesda, MD). binational and "SOS" repair. Recombinational processes are Preparation of Plasmid DNA. Plasmid DNA was purified thought to be error free. The dependence of UV-induced mu- either on a small scale (0.5 ml of culture) by the procedure of tations on the presence of a functional recA+ gene, however, Birnboim and Doly (8) or on a larger scale (1 liter ofculture) by has led to the hypothesis that there is a related SOS repair func- an adaptation ofthe procedures developed by Helinski and co- tion that is error prone. The recA+-dependent repair processes workers (9, 10). have been found to be induced by the presence of UV-damaged Modification of Plasmid DNA with UV Irradiation. Plasmid DNA-as well as by many other types of DNA damage. pKO482 DNA at a concentration of 20 jig/ml in 10 mM Tris- Although repair and mutagenesis have been studied for ge- HCVl10 mM CaCl2/10 mM MgCl2 (pH 7.0) was irradiated in nomic and phage DNA in E. coli, little is known about the ef- 1.0-ml portions in 54-mm-diameter Petri dishes with a germi- fects of the different repair systems of the host E. coli on plas- cidal lamp (15 W, Phillips). Radiant flux was measured with a mid DNA (1). We report here the results of studies using a UVX Digital Radiometer (Ultra-Violet Products, San Gabriel, system recently developed by Kakefuda and co-workers (4, 5) CA). The number ofthymine dimers formed per plasmid mol- in which plasmid DNA is modified in vitro with DNA-damaging ecule was calculated by using apreviously published conversion agents and then used to transform strains ofE. coli with variable factor (11) of0.041% thymine bases converted from 10 J/m2 of DNA-repair capacities. In vitro modification ofthe DNA allows UV. for an accurate quantitative assessment ofthe number ofdamage Modification of Plasmid DNA with N-AcO-AAF. N-AcO- sites per DNA molecule prior to exposure to in vivo repair sys- [3H]AAF (173 Ci/mol; 1 Ci = 3.7 x 1010 becquerels) was syn- tems. In addition, this system has the advantage of excluding thesized as described (12) from 2-nitro[ring-3H]fluorene (Com- any toxic effects of the DNA damaging agent on other cell pro- missariat a l'Energie Atomique, Saclay, France). The N-AcO- cesses or structures. [3H]AAF (42.5 ,uM) was then allowed to react for various

The publication costs ofthis article were defrayed in part by page charge Abbreviations: AAF, acetylaminofluorene; N-AcO-AAF, N-acetoxy-N- payment. This article must therefore be hereby marked "advertise- 2-acetylaminofluorene. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 4133 Downloaded by guest on September 25, 2021 4134 Genetics: Schmid et aL Proc. Natl. Acad. Sci. USA 79 (1982) lengths of time with plasmid DNA (50 Ag/ml) in 10 mM However, after transformation with pKO482, which contains Tris.HCV1 mM EDTA (pH' 8.0) containing 5% (vol/vol) a galK gene, these strains will normally form red colonies on ethanol. Removal ofunbound fluorene derivativeswas achieved MacConkey's agar due to restoration of the galactose metabo- by four successive ethanol precipitations. The number ofN-2- lism pathway and, consequently, production of an acidic envi- acetylaminofluorene molecules (AAF adducts) covalently bound ronment. A visually distinctwhite colony is formed ifa mutation per plasmid DNA molecule was determined by scintillation has occurred in the plasmid galK gene ofthe plasmid pKO482. counting of tritium radioactivity and measuring the nucleotide Verification ofthe presence of a plasmid galK gene mutation concentration from the absorbance at 260 nm. in thewhite colonieswas performedbyextraction ofthe plasmid Preparation and UV Irradiation of the Bacteria Prior to DNA as described above followed by (i) agarose gel electro- Transformation. Where indicated, suspensions ofE. coli in LB phoresis and (ii) retransformation into the AB 1157 strain to medium (2-4 X 108 cells per ml) were irradiated on ice with obtain only white colonies. Five percent ofthe more than 1,100 agermicidal lamp (15 W, Phillips). The dose ofUVwas adjusted mutants obtained in these experiments were randomly selected in each strain to give approximately 50% survival-i.e., 166 J/ for this procedure, and positive results were obtained in all m2 for AB 1157, 7.9 J/m2 for AB 1886, 2.3 J/m2 for AB 2463, cases. and 0.7 J/m2 for AB 2480. All subsequent procedures were car- Mutation frequency is expressed as the number ofwhite col- ried out under semidark conditions to minimize in vivo pho- onies pertotalnumberoftransformants. Thenumberofcolonies toreactivation of the thymine dimers. Cultures were next in- scored to obtain the mutation frequency for each experimental cubated for an additional 30 min at 370C to allow expression of condition and strain ranged between at least 20,000 for un- the SOS functions prior to the transformation step. These con- modified plasmid and at least 1,000 at the highest plasmid mod- ditions have been shown to be optimal for induction ofthe SOS ification levels reported. response in E. coli as measured by Weigle reactivation and mutagenesis in UV-irradiated phage A (13-15). RESULTS Transformation with Plasmid DNA and Selection for Trans- formed Cells. The E. coli were transformed with pKO482 by Relative Importance of uvrA+- and recA+-Dependent Re- the procedure of Mandel and Higa (16) as modified by Cohen pair Processes to Survival of Plasmid DNA Damaged by UV et aL (17). Selection for transformed cells was made by plating Irradiation or N-AcO-AAF Binding. Fig. 1 Left shows the de- on MacConkey's agar (18) containing 2% galactose and ampi- crease in survival ofplasmid pKO482 DNA containing increas- cillin at 50 Ag/ml and incubating overnight at 370C: the (3-lac- ing numbers ofthymine dimers when used to transform strains tamase gene of pKO482 provides ampicillin resistance to the of E. coli with different DNA repair capabilities. Thymine di- transformed cells. The transformation efficiency was found to mers are thought to be the major DNA lesion created by UV be a linear function of both plasmid and cell concentrations irradiation, although other less frequent types of UV lesions when the above conditions were used. Using 3 X 109 cells and cannot be excluded as lethal hits (1, 19). Our results show the 0.4 j.g ofplasmid DNA in afinal volume of 0.6 ml, we observed same relative effect ofhost cell recA and uvrA gene mutations the following approximate transformation efficiencies with un- on plasmid survival as reported by Roberts and Strike (20). modified plasmid: 5 x 105 colonies per pkg of DNA for the AB Comparison of the curve for the AB 2463 uvrt recA- strain to 1157 and AB 1886 strains, 2.5 X 105 colonies per ptg of DNA that for the AB 1157uvr' rec' strain shows that loss ofthe rec+- for the AB 2463 strain, and 0.2 X 105 colonies per 1Lg of DNA dependent DNA repair has little effect on total repair capacity for the AB 2480 strain. and hence the survival ofUV-irradiated plasmid. The same re- Detection, Verification, and Quantitation of Plasmid Mu- sult can be seen by comparing the AB 1886 uvrA- rec' strain tation. Because all ofthe bacterial strains used have a mutation to the AB 2480 uvrA- recA strain. Most ofthe repair to UV- in the genomic galK gene, they are unable to ferment galactose. damaged plasmid DNA is performed by host cell uvr'-depen-

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10 20 30 40 10 20 30 40 50 AAF adducts per plasmid molecule Thymine dimers per plasmid molecule FIG. 1. Survival of UV-irradiated (Left) or AAF adduct-containing (Right) plasmid pKO482 after it was used to transform E. coli strains with differing DNA repair capacities. The surviving fraction is calculated from the transformation efficiency by using plasmid DNA with the indicated number of thymine dimers per molecule relative to the transformation efficiency in the same strain when unmodifiedplasmid was used. The strains tested were AB 1157 uvr' rec' (o, *), AB 2463 uvr' recA (A, A), AB 1886 uvrA rec' (o, *), and AB 2480 uvrA recA- (o, *). Prior to transformation the cells had (solid symbols) or had not (open symbols) been irradiated with UV light to induce SOS functions. The curves for each strain represent the average of two to four experiments performed on.separate days. Downloaded by guest on September 25, 2021 Genetics: Schmid et al. Proc. Natl. Acad. Sci. USA 79 (1982) 4135 dent functions. This is demonstrated by the large decrease in rec+ strains, AB 1157 uvr+ rec+ and AB 1886 uvrA- rec+, are survival for UV-modified plasmid in the AB 1886 uvrA- rec' about the same. The frequencies found, 0.007 and 0.010, re- strain compared to the AB 1157 uvr' rec' strain and the AB spectively, are approximately 70-100 times greater than the 2480 uvrA- recA- strain compared to the AB 2463 uvr' recA- spontaneous mutation frequency ofapproximately 0.0001 found strain. for unmodified plasmid introduced into these strains by Fig. 1 Right shows the decrease in survival ofpKO482 DNA transformation. in each strain as a function of increasing modification of the N-AcO-AAF-modifted plasmid. The mutation frequencies in- plasmid with N-AcO-AAF. The relative effects on survival of duced by AAF adducts at the 1% survival level in the AB 1157 the uvr'- and rec'-dependent repair systems is quite similar and AB 1886 strains were 0.033 and 0.030, respectively (Fig. for AAF adducts and thymine dimers. As with thymine dimers, 2 Right and Table 1), which are at least 3 times greater than the the uvrA+-dependent excision repair process is also responsible frequencies found under comparable conditions (1% survival) for the majority ofrepair ofAAF adducts on plasmid DNA. This with UV-modified plasmid used to transform these two strains is in full agreement with our recent data showing that AAF- (Table 1). This higher mutation frequency for AAF adducts is modified DNA is incised in vitro by the uvr'-encoded endo- due at least in part to the presence ofadditional rec'-indepen- nuclease (unpublished data). reck-dependent repair is more im- dent mutagenesis as revealed by results in the two strains lack- portant to the survival ofN-AcO-AAF-modified than UV-mod- ing recA+ functions (AB 2463 and AB 2480). In contrast to the ified plasmid DNA but is still much less active than the excision results obtained with UV dimers (Fig. 2 Left), a measurable in- repair system in both cases. crease in the frequency of mutations was found in these two Relative Importance of the uvrA+- and recA+-Dependent strains as the number of AAF adducts per plasmid molecule Repair Processes to Mutations Induced in the Plasmid-En- increased (Fig. 2 Right). At the 1% plasmid survival level, the coded galK Gene. UV-damaged plasmid. Fig. 2 Left shows the galK gene mutation frequencies induced by AAF adducts in the frequency of forward mutation in the galK gene of plasmid recA strains (0.010 in AB 2463 and 0.006 in AB 2480) are sig- pKO482 used to transform each strain as a function ofthe num- nificantly greater than the spontaneous mutation frequency of ber of UV dimers per plasmid molecule. The strain deficient unmodified plasmid (approximately 0.0001). only in excision repair (AB 1886 uvrA- rec') has a significantly Induction ofthe SOS Functions Does Not Increase the Sur- higher frequency ofmutation at any given level of UV damage vival or the Mutagenesis of the Plasmid. To study the effect than does the wild-type strain (AB 1157 uvr' rec+). In the AB ofinducing the rec+-dependent SOS functions on plasmid sur- 2463 uvr' recA strain, deficient in the known error-prone vival and mutagenesis, in parallel experiments we UV-irradi- DNA repair system, the mutation frequency did not increase ated host cells prior to the transformation step. The conditions significantly above the spontaneous mutation frequency of used (see Materials and Methods) have been shown to be op- unexposed plasmid as the number of UV dimers increased. timal for induction of the SOS response in E. coli as measured Mutation frequency in the AB 2480 uvrA- recA double mutant by Weigle reactivation and mutagenesis in UV-damaged phage strain (not shown) could be assessed only at low levels ofplasmid A (13-15). However, our studies show that in the case for plas- modification due to the comparatively low transformation ef- mids, UV irradiation of rec' host strains does not increase the ficiency in this strain. However, at these low levels of modifi- survival (Fig. 1) or the mutagenesis (Fig. 2) of either AAF- or cation, mutation frequency was also found to remain at or near UV-modified plasmid DNA. This is true throughout the entire the spontaneous mutation frequency. range ofplasmid modification levels observed. In Table 1, comparison ofthe mutation frequency ofthe plas- mid-encoded galK gene in the various strains has been made DISCUSSION at equal levels ofsurvival of the plasmid (1%). When compared Howard-Flanders and Boyce (21) showed that excision (uvr+- under these conditions, the mutation frequencies in the two dependent) and postreplication (rec+-dependent) repair are

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10 20 30 40 50 60 10 20 30 40 Thymine dimers per plasmid molecule AAF adducts per plasmid molecule FIG. 2. Mutation frequency of the gaLK gene in plasmid pKO482 as a function of the number of thymine dimers (Left) or the number of AAF adducts (Right) per plasmid molecule in strains of E. coli with differing DNA repair capacities. The strains tested were AB 1157 uvr' rec' (o *), AB 2463 uvr' recA- (A, A), AB 1886 uvrA rec' (n, *), andAB 2480 uvrA recA - (see text). The cells had (closed symbols) or had not (open symbols) previously been irradiated with UV light to induce SOS functions. Downloaded by guest on September 25, 2021 4136 Genetics: Schmid et aL Proc. Natl. Acad. Sci. USA 79 (1982)

Table 1. Mutation frequency at 1% survival* for plasmid tribution of one type of repair to the total mutation frequency pKO482 containing thymine dimers or AAF adducts as seen in the wild type. We have chosen to compare mutation when used to transform E. coli strains with frequencies at the 1% survival level in Table 1 because this level differing DNA repair capabilities gave the best compromise between increasing mutation fre- quencies and decreasing survival from which to make the Transformed Mutation frequency calculations. strain With thymine dimers With AAF adducts The dependence of UV mutagenesis in phage (15, 26) and AB 1157 uvr' rec' 0.007 0.033 genomic (27, 28) DNA on recA+-dependent functions has been AB 2463 uvr' recA- <0.001 0.010 well substantiated. We have shown that, in the case for plasmid AB 1886 uvrA- rec' 0.010 0.030 DNA, the induction ofmutations by UV damage is also entirely AB 2480 uvrA recA <0.001t 0.006t dependent on recA+-dependent functions, because mutation * The mutation frequencies are taken from Fig. 2 at the level of plas- frequencyremained at the background level in both recA strains mid modification that gave 1% survival for each respective strain as studied (Fig. 1 and Table 1). Lack ofinduced mutation in UV- seen in Fig. 1. damagedplasmid in the AB 2463 uvr' recA strain, in particular, t Values given forthis strain are extrapolated from results at2.5% and also shows the error-free nature ofexcision repair, in agreement 4% survival for thymine dimers and AAF adducts, respectively, due with results reported for excision-dependent genomic DNA re- to a low transformation efficiency in this strain. pair (1, 3). The fact that the observed mutation frequencies (at equal survival levels) in the AB 1157 uvr' rec' and AB 1886 about equally important to the survival ofUV-irradiated E. coli uvrA rec' strains are not significantly different in the case for cells. The repair ofthe "bulky" adducts formed by the reaction either UV or N-AcO-AAF damage (Table 1) also shows the in- ofbenzo[a]pyrene-7,8-dihydrodiol-9, 10-oxide isomer I or 4-ni- dependence of mutation from uvr'-dependent functions. troquinoline-l-oxide with genomic DNA also appears to be In the case ofAAF-induced mutagenesis, a greater mutation about equally dependent on the two types ofrepair (22). In con- frequency was observed in recA+-proficient strains than in trast to the repair ofgenomic DNA, plasmid DNA is almost to- recA+-deficient strains at 1% survival (Table 1), showing that tally dependent on excision repair processes, at least in the case recA+-dependent error-prone repair does occur. However, be- for bulky lesions. Our results agree with those reported by Rob- cause a lower but significant level ofAAF-induced mutagenesis erts and Strike (20) for UV-damaged plasmid DNA, showing also occurs in recA strains, a second recA+-independent mu- almost complete lack ofrepair activity in uvrA host strains (Fig. tagenic process must exist to deal with AAF adducts. According 1 Left). We have shown that the repair of bulky AAF adducts to the SOS hypothesis (29, 30), the error-prone rec'-dependent on plasmid DNA also depends largely on excision processes functions are necessary when the DNA replication machinery (Fig. 1 Right). Using the same experimental system as reported is stopped by a lesion on the DNA template. When this hap- here, Mizusawa et aL (5) obtained similar results with benzo- pens, rec'-dependent processes are necessary to permit "read- [a]pyrene-7,8-dihydrodiol-9,10-oxide-modified plasmid. through" ofthe lesion, but with relaxed base pairing accuracy. We assume that plasmid survival depends on the ability of The bulk of a DNA adduct alone has been considered a factor the plasmid to be repaired and replicated by host mechanisms. in inhibition ofDNA polymerase (30). Bulky DNA adducts such By measuring the survival ofdamagedplasmid DNA in bacterial as UV dimers (our data; refs. 15 and 26), benzo[a]pyrene ad- strains deficient in certain repair processes, it is possible to as- ducts (5, 22), 4-nitroquinoline adducts (22), and large alkyl sess the relative importance ofthese processes in plasmid DNA groups (31, 32) are all completely dependent on rec+ functions repair. The survival curves in ourexperiments in some instances to induce mutagenesis. On the other hand, DNA alkylated with were nonlinear (non-log-linear), but when comparisons among methyl groups (22, 31, 32) gives both rec+-dependent and rec+- strains are made at equal survival levels throughout the mea- independent mutagenesis. In this sense, mutagenesis induced sured range the relative differences remain nearly constant. The by the large AAF adduct more closely resembles that induced basis for nonlinear survival curves is not yet clear. Similar re- by the small methyl group than that ofother bulky adducts. sults have been reported by others (19, 20, 23). Possibly the The most frequently occurring (80%) lesion found in N-AcO- nonlinearity reflects changes in the rate of the enzymatically AAF-modified DNA is one in which AAF is bound to the C-8 catalyzed DNA repair processes as a function of the cellular of guanine (33). The resulting conformational change of the concentration of the DNA lesion (substrate concentration). At DNA secondary structure is an anti-to-syn rotation that brings high concentrations of the lesion, the enzymatic repair system the modified guanine outside the helix while inserting the flu- would become saturated (Vm. reached), and only then would orene ring between the neighboring base pairs. This confor- the survival curves become truly linear. mational change, described as the "insertion-denaturation" Poisson statistics predicts that, at a given level of survival in model (34, 35), probably results in a noncoding lesion requiring two different host strains, the remaining average number ofle- rec+-dependent functions to be bypassed. Kadlubar (36), how- sions per plasmid molecule is the same in each (24, 25). The ever, has recently proposed a model for direct base mispairing average number ofadducts per molecule repaired by a certain (transversion mutations) by a less frequently occurring (10%) repair system at that survival level is the difference in average lesion in which AAF is bound to the N2 ofguanine. Our finding number ofadducts per molecule read from the survival curves of additional rec+-independent mutagenesis for AAF adducts for two strains that are isogeneic except for mutation in the given supports this hypothesis of direct base mispairing by a bulky repair system ofone. At equal survival, the number ofadducts adduct. Thus, inhibition ofthe replication process by a modified repaired (per molecule) by the uvr+-dependent system (AB base would appear to depend more on the lack ofbase-pairing 2463 strain minus AB 2480) plus the adducts repaired by the ability ofthe lesion (correct or incorrect pairing) than on adduct rec+-dependent system (AB 1886 minus AB 2480) should equal size. The sequence analysis of the mutations induced by AAF the number of adducts repaired in the wild-type strain con- in a recA host cell can be investigated by using a technique that taining both systems (AB 1157 minus AB 2480). We found this we have recently developed (37). Results ofsuch work may pro- relationship to hold throughout the measured range of survival vide more evidence as to the mechanism of rec'-independent levels with both UV- and N-AcO-AAF-modified plasmid DNA mutagenesis in AAF-modified DNA. (data not shown). Mutation frequencies compared between Both rec'-dependent misrepair and direct mispairing are strains at equal survival levels will also reflect the actual con- believed to occur at the replication fork, although it has also Downloaded by guest on September 25, 2021 Genetics: Schmid et aL Proc. Natl. Acad. Sci. USA 79 (1982) 4137 been suggested that these processes may operate during pre- 5. Mizusawa, H., Lee, C. & Kakefuda, T. (1981) Mutat. Res. 82, replicative excision repair in the case in which there is a second 47-57. lesion in the template strand within the length of the excision 6. Howard-Flanders, P., Boyce, R. P. & Theriot, L. (1966) Genetics 53, 1119-1136. patch (38). However, we must also consider the existence of 7. McKenney, K., Shimatake, H., Court, D. & Rosenberg, M. other mutagenic mechanisms that fix the mutation in both DNA (1980) Fed. Proc. Fed. Am. Soc. Exp. Biol. 39, 2203 (abstr.). strands at the prereplicative stage. Due to the multicopy state 8. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. and to the recessiveness of the mutation of this plasmid system 9. Katz, L., Kingbury, D. T. & Helinski, D. R. (1973) J. Bacteriol. it is most likely that the mutations that are scored in this assay 114, 577-591. prior to replication. 10. Clewell, D. B. & Helinski, D. R. (1970) Biochemistry 9, 4428- are fixed in both strands 4440. Another important observation is that UV irradiation of the 11. Seeberg, E. & Strike, P. (1976)J. Bacteriol 125, 787-795. host cells prior to transformation did not increase the survival 12. Le1evre, J., Fuchs, R. P. P. & Daune, M. P. (1978) Biochemistry or the mutagenesis of either UV- or N-AcO-AAF-treated plas- 17, 2561-2567. mid DNA. This is in contrast to the well-documented Weigle 13. Defais, M., Caillet-Fauquet, P., Fox, M. S. & Radman, M. reactivation and Weigle mutagenesis phenomena observed for (1976) Mol. Gen. Genet. 148, 125-130. been 14. Radman, M. & Devoret, R. (1971) Virology 43, 504-506. phages (13-15, 26, 39-41). Weigle mutagenesis has also 15. Defais, M., Fauquet, P., Radman, M. & Errera, M. (1971) Vi- described in the case ofphage 4X174 DNA that had been sub- rology 43, 495-503. jected in vitro to limited depurination (25). For UV-damaged 16. Mandel, M. & Higa, A. (1970)J. Mol. Biol 53, 159-162. phage A, the mutation frequency in an induced rec' host strain 17. Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972) Proc. Natl Acad. has been found to be 5-25 times greater than in the same non- Sci. USA 69, 2110-2114. induced host (15, 26). The survival frequency is also at least 10 18. Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures (1953) (Difco times greater under optimal conditions of phage and host cell Laboratories, Detroit), 9th Ed., pp. 131-132. irradiation (14, 26). In our assay system, increases in survival 19. Witkin, E. M. (1966) Science 152, 1345-1353. or mutagenesis frequency of as little as 2-fold would have been 20. Roberts, R. J. & Strike, P. (1981) Plasmid 5, 213-220. apparent. In addition, increases in the background mutation 21. Howard-Flanders, P. & Boyce, R. P. (1966) Radiat. Res. Suppt frequency ofundamaged phage ofas great as 10- to 40-fold have 6, 156-184. been reported as a result of UV induction of host cells (42-44). 22. Ivanovic, V. & Weinstein, I. B. (1980) Cancer Res. 40, 3508-3511. type of mutagene- 23. Salaj-Smic, E., Petranovic, D., Petranovic, M. & Trgovcevic, Z. We found no induction ofthis "nontargeted" (1979) Mol Gen. Genet 177, 91-94. sis in our studies on plasmid DNA. 24. Campbell, R. C. (1974) Statisticsfor Biologists (Cambridge Univ. One possible explanation for the observed noninducibility of Press, London), 2nd Ed., pp. 303-308. SOS repair in the case for plasmid DNA is that the presence 25. Schaaper, R. M. & Loeb, L. A. (1981) Proc. Natl Acad. Sci. USA of damaged plasmid DNA in the host might itself induce SOS 78, 1773-1777. functions such that the effects of host cell induction by UV ir- 26. Miura, A. & Tomizawa, J. (1968) Mol. Gen. Genet. 103, 1-10. radiation are masked. However, this would not explain the lack 27. Witkin, E. M. (1969) Mutat. Res. 8, 9-14. 28. Sedgwick, S. G. (1975) Proc. Natl Acad. Sci. USA 72, 2753-2757. of UV induction of nontargeted mutagenesis in plasmid DNA 29. Radman, M. (1974) in Molecular and Environmental Aspects of or why the presence of damaged phage DNA in the host does Mutagenesis, eds. Prakash, L., Sherman, F., Miller, M., Law- not similarly induce the SOS functions. rence, C. & Tabor, H. W. (Thomas, Springfield, IL), pp. 128- Whether or not inducible error-prone repair operates on 142. bacterial DNA is not clear. Data obtained with the tif-1 mu- 30. Radman, M., Villani, G., Boiteux, S., Defais, M. & Caillet-Fau- tation in the recA gene (45-47) support the idea that error-prone quet, P. (1977) in Origins of Human Cancer, eds. Hiatt, H. H., repair ofgenomic DNA is inducible. In contrast, it has recently Watson, J. D. & Winsten, J. A. (Cold Spring Harbor Laboratory, been shown by Salaj-Smic et al. (23) that this induction phe- Cold Spring Harbor, NY), Book B, pp. 903-922. nomenon is largely irrelevant to the repair of UV-damaged bac- 31. Garner, R. C., Pickering, C. & Martin, C. N. (1979) Chem.-Biol Interact. 26, 197-205. terial DNA. 32. Yoshikawa, K., Nakadate, M., Watabe, T., Ishadate, M., Jr., & From this study it seems that the inducible component ofthe Kondo, S. (1980) Mutat. Res. 79, 319-325. rec+-dependent repair system plays no role in the survival or 33. Kriek, E., Miller, H. A., Juhl, V. & Miller, E. C. (1967) Bio- mutagenesis ofplasmid DNA. Why does this host cell function chemistry 6, 177-182. operate differently for plasmid versus phage DNA? The mo- 34. Fuchs, R. & Daune, M. 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