Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3982-3986, June 1989 Biochemistry Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli (plasmid mutagenesis/pSV2-gpt/homologous recombination/angelicin/deletions) FRANCES M. SLADEK*t, AGUSTIN MELIANt, AND PAUL HOWARD-FLANDERS§ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511 Communicated by Fred Sherman, February 21, 1989 (receivedfor review June 10, 1988)

ABSTRACT 4,5',8-Trimethylpsoralen (psoralen) plus yield of SOS-dependent mutations (15, 16), which tend to be near UV light produces interstrand crosslinks and monoad- base-pair substitutions (17-19). ducts in DNA, both ofwhich are mutagenic. InEscherichia coil, The repair of crosslinks appears to occur by a sequential crosslinks are incised by UvrABC excinuclease, an event that excision-recombination mechanism (20, 21). In vitro studies can lead to homologous recombination and repair. To deter- show that crosslinked DNA is incised by the UvrABC exci- mine whether UvrABC incision of crosslinks is a step in the nuclease (22, 23) so that an 11-base oligonucleotide containing path to mutagenesis as well as repair, the effect of DNA the crosslink is left attached to an intact DNA strand (24). They homologous to a target gene on a plasmid was determined. also show that RecA-mediated strand exchange can proceed pSV2-gpt DNA was treated with psoralen and transformed into past the crosslinked oligonucleotide (25) and that a second a pair of hosts: one was gpt', the other was A(gpt-ac)5. The round of incision by UvrABC can release the psoralen moeity DNA was extracted and transformed into a tester strain from the DNA (25, 26). In vivo studies support the finding that [A(gpt-lac)5] in which Gpt- mutations in the plasmid were incision leads to homologous recombination: under repressed scored. The results show that psoralen-induced mutations were conditions, psoralen-crosslinked A phage induce RecA- reduced to background levels by the presence of the gpt' dependent genetic exchanges with a prophage in Uvr' cells homolog in the host chromosome. Agpt hosts that were con- but not in Uvr- cells (27). stitutively induced for the SOS response yielded point muta- Since incision by UvrABC is a first step in the repair of tions, whereas noninduced hosts yielded almost exclusively psoralen crosslinks, we reasoned that it might also be a first large deletions. Since crosslinks were estimated to be respon- step in the path to mutagenesis by crosslinks. If this hypoth- sible for most of the mutations observed, we conclude that the esis is correct, then repair of incised crosslinks via homolo- premutagenic lesion of psoralen crosslinks is recombinagenic gous recombination would be expected to compete with and therefore very likely to be the product ofUvrABC incision. mutagenesis by those crosslinks. To test this, the production of4,5',8-trimethylpsoralen/NUV-induced mutations in a tar- Psoralens are photosensitizing compounds that are often get gene (gpt') on a plasmid (pSV2-gpt) was determined in used in chemotherapy and that can lead to mutations in a wide the presence (gpt') and the absence [A(gpt-Iac)5] of homol- variety oforganisms from Escherichia coli to man (1, 2). The ogous chromosomal DNA in Uvr' cells. The results show genotoxic effect of psoralens comes from the ability of these that crosslink-induced mutations were reduced to back- three-ringed aromatic compounds (furocoumarins) to inter- ground levels by the presence of the gpt' homolog. They calate into DNA and, upon excitation by near UV light indicate that the premutagenic lesion ofpsoralen crosslinks is (NUV, 320-400 nm), to undergo cyclobutane addition with the 5,6 double bond ofadjacent pyrimidines. The result is the recombinagenic and therefore very probably the product of formation of both monoadducts and interstrand crosslinks, UvrABC incision. primarily at 5'-TpA-3' sequences (3). It has been apparent for some time that psoralen/NUV MATERIALS AND METHODS treatments producing both crosslinks and monoadducts in- duce mutations in E. coli (4-8). However, it was only Media. GAB plates contained E medium (28) supplemented recently that crosslinks alone were proven to be mutagenic with 1.7% Bacto-agar, 0.4% glucose, 0.5% Casamino acids, and not just lethal. Zhen et al. (9) constructed a plasmid free 0.0002% thiamin, 0.0001% biotin, 0.01% 6-thioguanine of monoadducts which contained a single psoralen crosslink (Sigma), and 100 tkg of ampicillin per ml. Amp plates were in a given position. Upon transformation into excision- Luria plates (29) plus ampicillin (100 ,ug/ml). proficient (Uvr+) E. coli cells, the crosslinked plasmid Bacterial Strains and Plasmids. The strains used are de- yielded mutations at a relatively high frequency (3%). This scribed in Table 1. The Gpt- and Gpt' phenotypes were finding reinforces those of others (10, 11) who found base verified by growth and lack thereof, respectively, in the changes in psoralen-damaged vectors primarily at sites that presence of6-thioguanine. Gpt- is referred to as ThioGR. The favored crosslinking. plasmids used, pSV2-gpt and pBR-gpt (34), were maintained The mechanism by which psoralen crosslinks produce in E. coli AB2487 and prepared as described (35). They are mutations in E. coli is not known but it appears to require identical with respect to the E. coli-derived gpt+ fragment SOS functions (9, 12, 13). These are a set ofdamage-inducible and ampicillin resistance and are similar in size. cellular responses that lead to increased DNA repair and to mutagenesis by a variety of DNA-damaging agents (14-16). Abbreviations: NUV, near ultraviolet light; ThioGR, 6-thioguanine- Derepression of LexA, activation of RecA protease, and the resistant (Gpt-); AmpR, ampicillin-resistant. presence of the UmuC/D proteins are required for maximal *Present address: The Rockefeller University, 1230 York Avenue, New York, NY 10021. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge tPresent address: Yale University School of Medicine, New Haven, payment. This article must therefore be hereby marked "advertisement" CT 06510. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §Deceased, September 16, 1988.

Downloaded by guest on October 2, 2021 3982 Biochemistry: Sladek et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3983 Table 1. E. coli K-12 strains used Other Strain Relevant genotype markers* Origin FS301 recA85 1exA7l::Tn5 A(gpt-Iac)5 A P1:NO200 x KL788 FS305 recA85 lexA7l::Tn5 gpt' A KL226 x FS301 FS100 A(gpt-lac)5 B KL584 x pro' lac' derivative of KL226 x AB1157 (ref. 30) FS101 A(gpt-lac)5 umuCJ22::Tn5 B P1:GW2100 x FS100 FS105 gpt+ B KL226 x FS100 FS203 A(gpt-4ac)5 umuCl22::TnS A(srl-recA)7 C P1:GW2100 x FS200 FS201 A(gpt-4ac)5 umuC122::TnS C recA+ srl+ derivative of FS203 FS200 A(gpt-4ac)5 A(srl-recA)7 C KL584 x KM4104 N0200 recA85 srlC::TnlO D N. Ossannat KL788 recA441 lexA71::TnS A(gpt-4ac)5 E Ref. 31 KL226 Hfr gpt+ F Ref. 32 GW2100 umuC122::Tn5 G Ref. 33 KL584 Hfr A(gpt-4ac)5 H Ref. 31 KM4104 A(srl-recA)7 I K. McEnteet AB2487 recA13 G Ref. 30 *A, mtl-i str-31 sulA3 srlC::TnlO; B, F- ara-14 argE3 his-4 galK2 mtl-1 rpsL31 sup-37(am) thi-I tsx33 xyl-5; C, argA A(gal-bio)2134 lysA rpsL mtlA; D, HfrH? thi-i reCA1 Alac cps-3 malF55::TnS sulA::MudX(Cam) (Mu c+); E, as A but srl+; F, pit-10 relAl spoT) tonA22 T2R; G, as B but leu-6 lacYI A(gpt-proA)62; H, cysG303 relAI? spoTI? metBi; I, as C but lacX74. tU.S. Department of Agriculture, Beltsville, MD. tUniversity of California, Los Angeles, School of Medicine. Treatment of Plasmid DNA. Form I pSV2-gpt DNA (100 followed. Plasmid DNA was treated in vitro with psoralen ,ug/ml), in 10 mM TrisHCl, pH 8.0/1 mM EDTA, was and NUV at 0 or 0.75 kJ/m2 (or with angelicin and NUV at supplemented with 4,5',8-trimethylpsoralen (referred to as 0 or 7.2 kJ/m2) and used to transform an isogenic pair of E. psoralen) at 1 ,4g/ml by addition of1% (vol/vol) ofa saturated coli hosts: one was gpt+, the other was Agpt. One pair was solution in ethanol. After 30 min, the DNA was irradiated constitutively induced for SOS functions (lexA71: :TnS with NUV of predominantly 365 nm for 75 sec at 10 J/ recA85), the other was noninduced (lexA+ recA+). After a (m2-sec), as measured by a UV meter (J-221; Ultraviolet period of incubation, AmpR transformants were selected and Products, San Gabriel, CA). In a similar fashion, pBR-gpt the plasmid DNA was isolated and assayed for gpt- muta- was supplemented with angelicin (10 ,g/ml) and irradiated tions in Agpt tester cells by growth on 6-thioguanine and for 720 sec. Control (or unirradiated) DNA was treated with ampicillin (GAB plates). The Gpt+ phenotype is dominant. psoralen or angelicin but no NUV. The requirement for SOS functions for psoralen-induced Transformation of Bacteria and Assay for Gpt- Phenotype. mutagenesis was verified by reversion of the his4 allele in The host and tester cells were grown and prepared for transformation as described (51). In general, 3 and 20 ml of Eco-gpt' competent host cells were transformed with 1.5 and 10 jig of control and irradiated DNA, respectively. Competent tester cells (6 ml) were transformed with -3 ,ug (as determined by ethidium bromide staining ofan agarose gel) ofplasmid DNA isolated from the hosts. These amounts of DNA were less than saturating. After the heat shock, overnight incubation in rich, nonselective medium was such that at most 1-2 cell divisions occurred. The host cells were spread on Amp plates and incubated 24 hr at 37°C. Tester cells were spread on Amp and GAB plates (approximately 1-104 transformed and 10W-109 nontransformed cells per dish) and incubated for 30 hr. Select AmpR Plasmid Amplification and Isolation. The ampicillin- Isolate plasmid resistant (Amp ) colonies from the transformations into the Transform tester cells hosts were harvested and pooled for each set of conditions (300-19,000 colonies for irradiated samples and 1200-150,000 for controls) and resuspended in Luria broth plus ampicillin Select AmpRThioGR (50 ,g/ml) to about 108 cells per ml. In general, 500 ml ofeach (determine plasmid size) cell was allowed to cell at suspension undergo 2-3 divisions Absence Compare Presence 37°C before addition of chloramphenicol (100 ,g/ml) to of Homolog Mutation Frequency in: of Homolog amplify the plasmid DNA overnight. The cells were then harvested and the plasmid DNA was isolated as described FIG. 1. Experimental design to determine the effect of a gpt+ (37). Yields of 2-50 Ag of plasmid DNA per 500-ml culture (chromosomal) homolog on psoralen/NUV-induced mutagenesis in were obtained. a plasmid gpt gene. Eco-gpt+ (shaded) is a 1048- to 1063-base-pair fragment from the E. coli chromosome and includes 450 bases of the coding region of the gpt+ gene (solid) (38, 39). Agpt is A(gpt-lac)5, RESULTS resulting from a 140-kilobase (kb) deletion in the chromosome whose exact ends are not known. SOS-induced hosts (lexA71 recA85) were Reduction of Psoralen-Induced Mutations in the Presence of FS301 (Agpt) and FS305 (gpt+). Tester cells (Agpt umuC122) were the gpt+ Homolog. To determine the effect of a gpt+ (chro- FS203 (RecAl) or FS201 (RecA+). ThioGR = Gpt-. Not shown are mosomal) homolog on psoralen-induced mutagenesis in a noninduced hosts (lexA+ recA+)-FS100 (Agpt) and FS105 (gpt+)- plasmid gpt gene, the experimental design in Fig. 1 was and angelicin/NUV treatment of pBR-gpt (5.2 kb). Downloaded by guest on October 2, 2021 3984 Biochemistry: Sladek et A Proc. Natl. Acad. Sci. USA 86 (1989)

umuC' (FS100) but not umuCJ22::TnS (FS101) cells upon presence of 6-thioguanine was low from the gptI host treatment with psoralen and NUV (data not shown; see ref. (column 4: 4.6 x 10-4 vs. 4.8 x 1O-4 for the control) but 13). The umuCJ22::TnS allele in the tester strains ensured unexpectedly high from the Agpt host (55 x 10-4). Therefore, that the mutations were produced in the host cells. A RecA' the plasmid DNA from 10 ThioGR AmpR colonies was (FS203) and a RecA' (FS201) tester strain were used: no analyzed; all 10 had large portions deleted (Table 2, column significant difference was noted between them (see Table 2). 8). While some deleted plasmids were subsequently found The results of passing psoralen-damaged plasmid DNA under virtually every condition (Table 2, column 8, Table 3, through the SOS-induced hosts into the tester cells are column 6), psoralen-damaged DNA transformed into the presented in Table 2, top section. The data from four separate Agpt, noninduced host produced by far the greatest number. transformations into the tester cells are given; the transfor- Restriction analysis revealed five different size classes (2.3- mations into the hosts were performed a total of three times 4.2 kb vs. 5.3 kb for the parent) from all sets of conditions; (the two lines of data given for the RecA- strain are for the each class invariably lacked all or most of the E. coli gpt same preparation of DNA passed through the hosts). Col- fragment but had the gene for ampicillin resistance (data not umns 3 and 4 of Table 2 show that psoralen-induced muta- shown). tions were produced in the SOS-induced Agpt host (FS301) Relative Contribution of Psoralen Monoadducts and and successfully transferred to the tester cells (e.g., column Crosslinks to Mutagenesis: Comparison with Angelicin. To 3, top lines: 1.5 x i0- mutants per transformant from determine the relative contribution of monoadducts and unirradiated DNA vs. 16 x 10-4 from irradiated DNA). In crosslinks to psoralen-induced mutagenesis, the experiment contrast, there were no psoralen-induced mutations pro- was repeated with angelicin, an isomer of psoralen that duced upon passage of the DNA through the SOS-induced, produces only monoadducts (40). 4,5',8-Trimethylpsoralen gpt+ host (FS305) in that experiment (column 3, top lines: 2.2 monoadducts are not chemically equivalent to angelicin X 10-4 VS. 1.8 x 10-4). To take into account experimental monoadducts (3), but both undergo uvr-dependent excision variations in the number ofAmpR colonies pooled for each set repair (23, 41) and both appear to require SOS functions for of conditions and in the ThioGR AmpR colony counts, mutagenesis (42, 43). The results are presented in Table 3. weighted means of the mutation frequencies were calculated The weighted means suggest that angelicin-induced muta- (Table 2, columns 5 and 6). tions were reduced by a factor of =3 in the gpt' host (Table Psoralen-Induced Deletions in Noninduced Cells. To deter- 3, column 5: 6.9 x 10-4 for gpt' vs. 20 X 10-4 for Agpt). mine the fate of crosslinked DNA in the absence of SOS The psoralen-damaged DNA contained, on the average, 3 induction, the experiment was repeated with lexA+ recA+ interstrand crosslinks per plasmid molecule (as determined hosts (FS100 and FS105) (Table 2, middle section). The by alkaline gel electrophoresis of linearized plasmids as in psoralen-induced mutation frequency based on growth in the ref. 44) and 30-45 monoadducts (based on calculations as in Table 2. Frequency of psoralen/NUV-induced mutations in a plasmid gpt gene: Effect of a gpt' (chromosomal) homolog and SOS induction in the host Mutation frequency x 104 Fraction of NUV plasmids with Point mutation to DNA, Total Mean parental size frequency x 104 kJ/m2 Host RecA+ RecA- RecAl RecA- RecA+ RecA- RecA+ RecA- [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] Plasmid DNA passed through SOS-induced hosts and transformed into tester cells 0 Agpt 1.5 ± 0.6 11 ± 13 2.0 11 4/4 2/3 2.0 7.3 19 ± 44 0 0.75 Agpt 16 ± 7.2 50 ± 9.0 23 54 4/4 34/34 23 54 100 ± 148 60 ± 14 0 gpt+ 2.2 ± 1.0 4.5 ± 2.0 4.0 3.2 4/4 29/31 4.0 3.0 21±28 2.6±0.8 0.75 gpt+ 1.8 ± 0.8 12 ± 4.2 1.8 13 4/4 17/38* 1.8 5.8 0 29±44 Plasmid DNA passed through noninduced hosts and transformed into tester cells 0 Agpt 16 ± 4.8 5/10 8.0 0.75 Agpt 55 ± 24 - - 0/10 0 0 gpt+ 4.8 ± 2.8 - 5/9 2.6 0.75 gpt+ 4.6 ± 2.8 12/12 - 4.6 Plasmid DNA transformed directly into tester cells 0 0.34 ± 0.08 5.0 ± 2.0 11 9.5 3/4 23/26 8.3 8.4 27 ± 8.0 15 ± 6.4 0.75 0.7 ± 1.1 0.7 0 2/2 - 0.7 0 0 0 Results are from experiments as outlined in Fig. 1 and described in the text. All data are from transformations into tester cells. Column 1: 3 ,ug of pSV2-gpt DNA was used except for 0.5 ,ug for the unirradiated, direct transformation. Column 2: SOS-induced hosts were FS301 (Agpt) and FS305 (gpt+); noninduced hosts were FS100 (Agpt) and FS105 (gpt'). Columns 3 and 4: data from two transformations into each tester strain; RecA+ is FS201 and RecA- is FS203. Total mutation frequency is the number ofAmpR ThioGR colonies divided by the total number ofAmpR colonies. Errors are estimated 95% confidence limits based on 2 times the square root ofn, where n is the number of colonies counted on Amp and GAB plates and the number of AmpR colonies pooled for each transformation into the host. Columns S and 6: weighted mean of the two experiments for each tester strain. Columns 7 and 8: number of plasmids with parental size per total number analyzed from AmpR ThioGR colonies scored in the tester cells. The plasmids that were not the same size as the parent were smaller (deleted plasmids) in all cases except one (*) where one plasmid was larger. Columns 9 and 10: point mutation frequency was calculated using only that fraction of the ThioGR plasmids that were of parental size [i.e., column 10 = column 6 (or 4) x column 8]. Downloaded by guest on October 2, 2021 Biochemistry: Sladek et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3985 Table 3. Frequency of angelicin/NUV-induced mutations in a plasmid gpt gene: Effect of a gpt+ homolog in SOS-induced hosts Mutation frequency x 104 NUV Fraction of to DNA, Total plasmids Point mutation kJ/m2 Host Exp. a Exp. b Mean with parental size frequency x 104 [1] [2] [31 [4] [5] [6] [7] Plasmid DNA passed through SOS-induced hosts and transformed into tester cells o Agpt 3.2 ± 1.6 5.3 ± 3.8 3.9 7.2 Agpt 6.8 ± 2.4 29 ± 8.6 20 24/24 20 0 gpt+ 0.65 ± 0.6 3.0 ± 0.6 2.9 7.2 gpt+ 3.8 ± 0.8 7.3 ± 2.8 6.9 49/54 6.3 19 ± 7.6t Plasmid DNA transformed directly into tester cells 0 0.94 ± 0.14 2.3 ± 1.4 1.0 7.2 0 0 Results of experiments as outlined in Fig. 1 and described in the text. Notes are as in Table 2 except that pBR-gpt DNA was used and that experiments a and b were both done in the RecA- tester strain, FS203; the data in column 6 are from both experiments. See columns 8 and 10 of Table 2 for notes on columns 6 and 7. tData from a repeat transformation into tester cells of the irradiated DNA isolated from the gpt + host. ref. 36). The angelicin-damaged DNA contained an estimated in the chromosome of the SOS-induced host greatly reduced 120 monoadducts per plasmid molecule based on parallel the yield of point mutations in DNA containing psoralen studies with 3H-labeled 5'-methylangelicin (HRI Associates, crosslinks. Emeryville, CA). Assuming that psoralen monoadducts are equivalent to angelicin monoadducts with respect to muta- genesis, a comparison of the yields of induced mutations (54 DISCUSSION vs. 20 x 10-4, psoralen vs. angelicin) would suggest that, per The results show that psoralen/NUV-induced point muta- adduct, the crosslink could be as much as 100 times more tions in a gpt+ gene on a plasmid were produced in SOS- mutagenic than the monoadduct. We therefore conclude that induced E. coli hosts that carried A(gpt-lac)5 in their chro- crosslinks were the major contributor to mutagenesis in our mosome but not in those carrying gpt+. They also indicate psoralen experiments. Another study (11) reached a similar that the majority of the mutations observed were due to conclusion. interstrand crosslinks. The possibility that psoralen-induced Psoralen- and Angelicin-Induced Point Mutations in SOS- the Induced Hosts. Since we were interested primarily in the mutagenesis in general is blocked by the presence of gpt- point mutations that are characteristic ofthe SOS response to lac portion of the E. coli chromosome was ruled out by damaged DNA (17-19), we calculated the number of ThioGR verifying that the gpt+ and the Agpt SOS-induced hosts were AmpR mutants that harbored plasmids of parental size and equally mutable by psoralen at a separate (chromosomal) called them point mutations. The results are presented in locus, rpoB (data not shown, see ref. 13). We conclude that columns 9 and 10 of Table 2 and column 7 of Table 3; Fig. 2 mutations induced by psoralen crosslinks are reduced by the is a graph of the same data. The result is clear: as predicted presence of DNA homologous to the target gene and there- from our initial hypothesis, the presence ofthe gpt+ homolog fore by repair via homologous recombination. Lin et al. (27) showed that psoralen crosslinks induced NON- recombination in Uvr+ cells but not in Uvr- cells. While that INDUCED 606INDUCED.--a b;' I(C:: result was obtained under conditions in which the crosslinked R\i lso~iAi- 4 h^M;-;:I2A (A) DNA was not replicating, it is likely that UvrABC incision (bin) (C is also required for recombination under conditions permis- -to 50 t- sive to replication, such as in our experiments. The alterna- x tive would require an enzymatic function other than UvrABC to break the covalent bond of the crosslink. Such activities z 40.- LL have been proposed (45), but there is little ifany evidence that D they exist. c3L1 30 Support for the hypothesis that UvrABC incision is nec- LL essary for psoralen crosslink-induced mutagenesis was given zm 0 by Igali et al. (5), who noted that 8-methoxypsoralen pro- duced tryptophan revertants in Uvr+ but not Uvr- E. coli. F- Yatagai et al. (46) observed mutations in uvrB- cells treated with 8-methoxypsoralen, but none were at crosslinkable sites. Two groups (6, 7) also found 8-methoxypsoralen- induced mutations in Uvr- cells, but when the cells were reirradiated with NUV to convert monoadducts to cross- t , |- \ N % t \ .\ -- links, the yield of mutations decreased. Since cell survival NUV - t ;------+ I also decreased upon reirradiation, it was concluded that FIG. 2. Frequency of psoralen- and angelicin-induced point crosslinks were lethal but not mutagenic in E. coli (6, 7). mutations in a plasmid gpt gene: effect of the presence of a gpt' Now, it is apparent that crosslinks are mutagenic, but ap- homolog and SOS induction. Results are from experiments as parently only in Uvr+ cells. [We tried, unsuccessfully, to outlined in Fig. 1 and as presented in column 10 of Table 2 and obtain psoralen-induced mutations in pSV2-gpt in a uvrA- columns 5 (- NUV) and 7 (+ NUV) of Table 3. strain (AB1886, ref. 47) preirradiated with far UV to induce Downloaded by guest on October 2, 2021 3986 Biochemistry: Sladek et A Proc. Natl. Acad. Sci. USA 86 (1989) SOS: no ThioGR AmpR colonies were observed out of 2100 berger, M. (1980) Nature (London) 285, 407-409. AmpR transformants assayed.] 9. Zhen, W.-P., Jeppesen, C. & Nielsen, P. E. (1986) Photochem. The results from our angelicin experiments also suggest Photobiol. 44, 47-51. 10. Saffran, W. A. & Cantor, C. R. (1984) Nucleic Acids Res. 12, that the premutagenic lesion of angelicin monoadducts is 9237-9248. recombinagenic. Psoralen monoadducts are recombinagenic 11. Piette, J., Decuyper-Debergh, D. & Gamper, H. (1985) Proc. in Uvr- cells (27) but not very recombinagenic in nonrepli- Nati. Acad. Sci. USA 82, 7355-7359. cating DNA (36). Therefore, we propose that monoadducts 12. Saffran, W. A. & Cantor, C. R. (1984) J. Mol. Biol. 178, 595- become mutagenic primarily when they remain in the DNA 609. and block DNA synthesis to produce postreplication daugh- 13. Sladek, F. M. (1988) Ph.D. Dissertation (Yale Univ., New Haven, CT). ter-strand gaps. A gap in only one sister chromatid, however, 14. Witkin, E. M. (1976) Bacteriol. Rev. 40, 869-907. is likely to be repaired via homologous recombination with 15. Walker, G. C. (1984) Microbiol. Rev. 48, 60-93. the other, intact chromatid, as has been found with far UV 16. Peterson, K. R., Ossanna, N., Thliveris, A. T., Ennis, D. G. & light-induced damage (48, 49). Therefore, monoadducts prob- Mount, D. W. (1988) J. Bacteriol. 170, 1-4. ably induce mutations only when the sister duplex is not 17. Miller, J. H. (1982) Cell 31, 5-7. intact, such as when lesions are so closely spaced that they 18. Miller, J. H. (1983) Annu. Rev. Genet. 17, 215-238. 19. Foster, P. L., Eisenstadt, E. & Cairns, J. (1982) Nature cause overlapping gaps in opposite nascent DNA strands. A (London) 299, 365-367. similar model was proposed for mutations induced by far UV 20. Cole, R. S. (1973) Proc. Natl. Acad. Sci. USA 70, 1064-1068. light (50). 21. Sancar, A. & Sancar, G. (1988) Annu. Rev. Biochem. 57, 29- The noninduced Agpt host produced a relatively high 67. frequency of large deletions in the psoralen-damaged DNA. 22. Seeberg, E. (1981) Mutat. Res. 82, 11-22. 23. Sancar, A., Franklin, K. A. & Sancar, G. (1985) J. Mol. Biol. This result may have been exaggerated by the presence of 184, 725-734. several kilobases of nonessential DNA. Treatment of the 24. Van Houten, B., Gamper, H., Holbrook, S. R., Hearst, J. E. plasmid with far UV light, however, did not produce dele- & Sancar, A. (1986) Proc. Natl. Acad. Sci. USA 83, 8077-8081. tions in the noninduced host (data not shown), suggesting that 25. Sladek, F. M., Munn, M. M., Rupp, W. D. & Howard-Flan- the deletions might be specific to crosslink repair. After ders, P. (1989) J. Biol. Chem. 264, 6755-6765. incision by UvrABC, one DNA strand contains a crosslinked 26. Cheng., S., Van Houten, B., Gamper, H. B., Sancar, A. & oligonucleotide while the other contains, in essence, a gap. Hearst, J. (1988) J. Biol. Chem. 263, 15110-15117. 27. Lin, P.-F., Bardwell, E. & Howard-Flanders, P. (1977) Proc. Therefore, a cell that is not capable of mutagenic (SOS) Natl. Acad. Sci. USA 74, 291-295. bypass of the crosslinked oligonucleotide has no choice but 28. Vogel, H. J. & Bonner, D. M. (1956) J. Biol. Chem. 218, 97- to save the other, gapped strand, which probably would have 106. undergone nuclease digestion. While others (11) found point 29. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold mutations upon transformation ofa psoralen-damaged vector Spring Harbor Lab., Cold Spring Harbor, NY). into noninduced cells, their system was based on bacterio- 30. Bachmann, B. J. (1972) Bacteriol. Rev. 36, 525-557. phage M13 and could not detect large deletions. They also 31. Miller, J. H. & Low, K. B. (1984) Cell 37, 675-682. was induced the 32. Low, B. (1973) J. Bacteriol. 113, 798-812. concluded that the SOS system by damaged 33. Elledge, S. J. & Walker, G. C. (1983) J. Mol. Biol. 164, 175- vector. Ifinduction took place in our system, it did not result 192. in an increase in point mutations. 34. Mulligan, R. C. & Berg, P. (1980) Science 209, 1422-1427. In conclusion, the presence ofDNA homologous to a target 35. Godson, G. N. & Boyer, H. (1974) Virology 62, 270-275. gene reduced mutations induced by psoralen crosslinks to 36. Cassuto, E., Gross, N., Bardwell, E. & Howard-Flanders, P. background levels, indicating that recombinational repair (1977) Biochim. Biophys. Acta 475, 589-600. competed with the process of mutagenesis. Since UvrABC 37. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, 193- renders crosslinks we 197. incision psoralen recombinagenic, 38. Nuesch, J. & Schumperli, D. (1984) Gene 32, 243-249. conclude that incision by UvrABC is a step in the pathway to 39. Jagadeeswaran, P., Ashman, C. R., Roberts, S. & Langenberg, mutagenesis by those lesions. J. (1984) Gene 31, 309-313. 40. Ashwood-Smith, M. J. & Grant, E. (1977) Experientia 33, 384- F.M.S. and A.M. dedicate this paper to the memory of Paul 386. Howard-Flanders for his outstanding work in DNA repair. This work 41. Grossweiner, L. I. & Smith, K. C. (1981) Photochem. 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