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Incision by Uvrabc Excinuclease Is a Step in the Path to Mutagenesis By 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.
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