Duplication Mutation As an SOS Response in Escherichia Coli
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Copyright 0 1989 by the Genetics Society of America Duplication Mutation as anSOS Response in Escherichia coli: Enhanced Duplication Formationby a Constitutively Activated RecA Joan Dimpfl and Harrison Echols Department of Molecular Biology, University of CaE$ornia, Berkeley, California 94720 Manuscript received March 10, 1989 Accepted for publication July 6, 1989 ABSTRACT The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes withthe presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system producesa large increasein the frequency of point mutations. To examine the possibility that other types of mutations are induced as partof the SOS response, we have studied the production of tandem duplications.To avoid the complications of indirect effectsof the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway.The induction of pathways for both duplica- tions and point mutationsshows that the SOS system produces a broad mutagenic response.We have suggested previously that many typesof mutations might be inducedby severe environmental stress, thereby enhancing genetic variationin an endangered population. HE introduction of a replication-inhibitinglesion deletions, translocations) (ECHOLS198 1 ; 1982; Mc- T (by UV light or a chemical mutagen) into the DONALD1984). Indeed, UV light and chemical mu- DNA of Escherichia coli results in the induction of a tagens increase the frequency of duplications (HILL multioperon regulatory system which rescues the bac- and COMBRIATO1973; HOFFMANNet al. 1985). To terialpopulation from its threatened demise (SOS investigate whether this duplication mutagenesis is a response)(RADMAN 1975; WITKIN 1976; ECHOLS direct consequence of SOS induction, we have exam- 198 1; WALKER 1984).The SOS response is normally ined the effect of a RecA protein constitutively acti- turned on by the activation of the RecA protein to vated by mutation for the SOS response in the absence mediate cleavage of the LexA repressor of SOS ope- of external treatment (RecA730). rons (LITTLEand MOUNT 1982). This activation is Tandem gene duplications are an importanttype of probablyaccomplished by the association of RecA mutation in E. coli, occurring at high frequency with single-strandedDNA (ROBERTSand DEVORET to lo-’ per cell) and often including large segments 1983), or distortions in double-stranded DNA gener- of the chromosome (ANDERSONand ROTH 1977; ated by the DNA lesions (Lu, SCHEUERMANNand PETESand HILL 1988). These frequent large dupli- ECHOLS1986; Lu and ECHOLS1987). Many of the cations are primarilyRecA-dependent events, with induced genes code for proteins involved in excision endpoints corresponding to regions of homology.Ac- and recombinational repair, but two genes, umuC and cording to one model, unequal crossover events at umuD, are specifically required for a large, SOS-de- regions of homology might lead to tandem copies of pendent increase in the frequency of point mutations the intervening genes, via RecA-mediated recombi- (WALKER1984, 1985). Thus, the SOS response can nation (ANDERSONand ROTH 1977; PETESand HILL be considered to have two general features favoring 1988). Duringthe emergency state ofSOS, additional survival of the stressed bacterial population: increased duplications might occurby this mechanism (e.g.,from capacity for DNA repair and enhanced geneticvaria- overproduction of RecA) or by others (e.g., recombi- tion (RADMAN 1980;ECHOLS 1981, 1982). national repair around lesions). We have studied the The genetic variation hypothesis for SOS mutagen- effect of a constitutive SOS response on duplications esis suggests that other types of mutations might also at several loci, using two measurementsbased on gene be inducible (e.g., tandem duplications,inversions, dosage. The first assay measures duplications of the glyS gene. The glyS gene lies within a segment of DNA The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “oduertisement” that is highly susceptible to duplication (approximately in accordance with 18 U.S.C. 91734 solely to indicate this fact. per cell), presumably because of two large ho- Genetics 123: 255-260 (October, 1989) 256 J. Dimpfl and H. Echols Segregation analysis of duplications: These measure- ments were based on techniques developed for Salmonella + c (ANDERSON,MILLER and ROTH 1976). To remove selective rhsB e pressurefavoring glySL duplications,individual colonies weregrown at 37" inLB medium containing0.1 mM tryptophan for 10 to15 generations before plating logphase cultures for individualcolonies on the original selective medium lacking tryptophan. Similarly, to remove selective rhsB glyS xyl A > 13 glyS xyl rhsA rntl pressure favoring pur duplications, isolated colonies were grown at 37" in LB medium with 5 mM adenine. Loss of the duplication was assayed by replica plating to score loss FIGURE1 .-Map of the glyS region in Escherichia coli (after LIN, of either tetracycline resistanceor adenine prototrophy. CAPACEand HILL1984). The glyS gene is flanked by homologous regions B and A. Unequal crossover events between daughter chromosomes could result in tandem copies of glyS. RESULTS mologous regions (rhsB and rhsA) flanking the 140- Induction of gZySL duplications by introduction of kbp segment (Figure 1) (LIN,CAPAGE and HILL 1984). recA730: Of the two assays used to measure duplica- We have found that thefrequency of glyS duplications tions, we have selected the glySL system as the method rises about ten-fold when a wild-type recA gene is ofchoice because the measurements are the most replaced with the recA730 allele. We have also used a direct and have given the most consistent results. second type of measurement basedon transduction of Duplications of the glySL gene are recognized as large an insertion mutation into a recipient strain (ANDER- colonies after growth under low temperature condi- SON, MILLER and ROTH 1976). Using this assay, we tions. To determine the effect of a constitutive SOS confirmed the occurrenceof induced duplication for- response on the frequency of glySL duplication, we mation at the purC, purE and PurF genes. Based on introduced recA730 and sulA- (to prevent filamenta- additional genetic analysis, the induced pathway for tion) into the glySL trpA36 glyU (Sup) background duplication mutations appears to be distinct from the (GEORGE, CASTELLAZIand BUTTIN 1975; WITKIN et pathway for point mutationsand also differs from the al. 1982). We then compared the relative frequency major normal recombination pathway. of large colonies with that found in strains with wild- type recA after growth under selective plating condi- MATERIALS AND METHODS tions (Table 2). Strains with wild-type recA yielded a Bacterial strains: Bacterial strains are listed in Table 1 frequency of largecolonies of approximately 10-4, in with their genotypes. New strains were prepared using P1 agreement with previous reports (CAPAGEand HILL transduction of transposon insertionsin or near the gene of 1979; LIN, CAPAGEand HILL 1984). Two independ- interest (MILLER 1972). ent isolates containing recA730, however, produced a Assay of gZy& duplications: These measurements were ninefold increase in frequency of large colonies, ap- performed essentially as described (LIN,CAPAGE and HILL 1984). The selection method requires a cold-sensitive mu- proaching lo-' (Table 2, lines 2 and 3). From these tant glycyl-tRNA synthetasegene, glySL, in conjunction with data, we conclude that aconstitutively activated RecA trpA36 glyU (Sup) (FOLKand BERG 197 1; CAPAGEand HILL protein is sufficient for an increase in putative glyS 1979; LIN, CAPAGEand HILL1984). At 20", the glycine- duplication events, and that the introduction of rep- inserting missense suppressors are charged more efficiently lication-blocking lesions by external treatment is not in a glySL duplication mutant, leading tobetter growth and large colony formationin the absence of tryptophan. South- necessary for this increase. ern blot analysis has shownthat this selection methodis very Segregationexperiments: Large colonies repre- specific for duplications ofthe glySL gene (LIN,CAPAGE and senting possible glyS duplication events could also be HILL1984). In our assays, cells in log phase growth were due to mutations overcoming the inefficient suppres- plated at 20" and 37" on minimal plates supplementedwith sion of the trpA36 missense mutation (although the glucose, casamino acids and thiamine. At day 4, the 20" plates were examinedfor large colonies, representing poten- glySL test shows good correlation between large colony tial duplications of the glySL gene. The expression of the size and glySL duplications in a wild-type recA strain). SOS pathway was tested by a plaque assay for cleavage of For example, reversion of trpA36 to wild-type &PA, the X CZ repressor (MOUNT1977). mutations at the gZyU gene, or mutations increasing Assay of pur duplications: Duplications inpurine synthe-