Control of Large Chromosomal Duplications in Escherichia Coli by the Mismatch Repair System

(:opyright 0 199 I by the Genetics Society of America Control of Large Chromosomal Duplications in Escherichia coli by the Mismatch Repair System Marie-Agnes Petit,* Joan Dimpfl,* Miroslav Radmant and Harrison Echols* “Division of Biochemistry and Molecular Biology, University of Calfornia, Berkeley, Calfornia 94720, and ‘Institut Jacques Monod, F-75251 Paris-Cedex 05, France Manuscript received May 23, 199 1 Accepted for publication July 2, 1991 ABSTRACT Excessive recombination between repeated, interspersed, and divergedDNA sequences is a poten- tial source of genomic instability. We have investigated the possibility that a mechanism exists to suppress genetic exchange between these quasi-homologous (homeologous) sequences. We examined the role of the general mismatch repair system of Escherichia coli because previous work has shown that the mismatch repair pathway functions as a barrier to interspecies recombination between E. coli and Salmonella typhimurium. The formation of large duplications by homeologous recombination in E. coli was increased some tenfold by mutations in the mutL and mutS genes that encode themismatch recognition proteins. These findings indicate that the mismatch recognition proteins act to prevent excessive intrachromosomal exchanges. We conclude that mismatch repair proteins serve as general controllers of the fidelity of genetic inheritance, acting to suppress chromosomal rearrangements as well as point mutations. SCHERICHIA coli and Salmonellatyphimurium creased genetic variation for an endangered bacterial E carry large duplications of genomic segments at population (DIMPFLand ECHOLS1989). Based on the frequenciesthat can be as high as of abacterial SOS work, we were led to explore mechanisms that population (ANDERSONand ROTH 1977; PETESand might normally suppress formation of large duplica- HILL1988). These duplications are thought to arise tions and other chromosomal rearrangements. by an unequal recombination event between homol- A property of the rrn and rhs gene families is a ogous sequences dispersed in the genome. Two ex- slight divergence in DNA sequence(homeologous amples of such “gene families” are therrn operons for sequences) (HILL, HARVEYand GRAY1990). We were ribosomal RNA andthe rhs sequences (ANDERSON intrigued with the possible regulatory significance of and ROTH 1977; PETESand HILL 1988). Seven rrn this property because the recombinational barrier be- loci are present in the E. coli chromosome.More tween the homeologous DNA sequences of E. coli and recently, up to five rhs loci have been described in E. S. typhimurium can be largely eliminated by mutations coli; the rhs sequences lack a known genetic function in the mismatch repair system (RAYSSIGUIER,THALER (SADOSKYet al. 1989; FEULNERet al. 1990). In addition and RADMAN1989). Thus, mismatch repair proteins to duplication, rrn operons have been defined as sites might function aspart of a generalmechanism to limit for inversion, deletion, and transposition of chromo- intrachromosomal as well as interchromosomalre- somal segments (HILLet al. 1977; HILLand HARNISH combination between duplicated, diverged DNA se- 1981, 1982). quences. Chromosomalrearrangements, especially duplica- To study the recombination events leading to du- tions, can serve as valuable sources for environmental plication, we have used a bacterial strain that allows or evolutionary adaptation. For example, duplication quantitative measurementof duplications between the in Salmonella appears to aid growth on limiting car- rhsA and rhsB sequences some 140 kbp apart in the bon source, probably by a gene dosage effect (SONTI genome (LIN, CAPAGEand HILL1984). The rhsA and and ROTH 1989).However, some geneticre- rhsB sequences share a 3.7-kbp region of substantial arrangements, especially deletions, can be highly del- homology, as judged by hybridization data, but differ eterious. Recent work has shown that the frequency by 22 mismatches among the 990 bp alreadyavailable of large duplications in E. coli increases tenfold when for sequence comparison (SADOSKYet al. 1989; FEUL- the SOS response is constitutively activated (DIMPFL NER et al. 1990; C. W. HILL,personal communication). and ECHOLS1989). This observation indicates that The formation of largeduplications was increased duplication formation might be subject to some form some tenfold by mutations inactivating the mutL and of cellular regulation; the SOS response might allow mutS genes of the mismatch repair pathway. Thus, morefrequent rearrangements as a source of in- mismatch repairproteins are likely to function in Genetics 129 327-332 (October, 1991) 328 M.-A. Petit et al. MATERIALSAND METHODS for details). A schematic map of the CHI 504 chromosome, before and after duplication, is presented in Figure 1. Duplication frequency was measured in strain CH 1504 and its deriv- atives harboring a mutL, mutS, mutH or uvrD mutation. Each of these mutations inactivates the general mismatch repair pathway (MODRICH 1989; RADMAN FIGURE1 .-Chromosome map of strains used to detect duplica- 1989). The results of duplication assays are presented tion events. The structureof the duplicated chromosome is depicted in Table 2. In a mut+ context, the duplication fre- on the right side of the figure, with the arrows representing the quency was 1.1 X 1 0-4, which agrees with previously rhsA and rhsB sequences. reported data (LIN, CAPACEand HILL 1984; DIMPFL limiting intrachromosomal exchanges. Additional ex- and ECHOLS1989). In the absence of an active mutL periments indicate that the recombination events in- or mutS gene product, duplication frequency was 10- hibited by the MutL and MutS mismatchproteins are 15-fold higher (Table 2, lines 2 and 3). In contrast, RecBC-dependent. the mutH and uvrD mutants did not show an increase in duplication frequency (Table 2, lines 4 and 5). MATERIALS AND METHODS To verify the presence of a duplication in the large colonies, a segregation experiment was performed on Bacterial strains: Bacterial strains are listed in Table 1 several purified colonies. Duplication mutations are with their genotypes. New strains were prepared using trans- distinguished by their instability when the selective duction by phage P1 of transposon insertions in the gene of interest. pressure for them is removed, presumably because of Assay of gly& duplications: The genetic test allowing homologous recombination within the duplicated re- the detection of duplications has been previously described gion (CAMPBELL1965; ANDERSONand ROTH 1977). (LIN, CAPACEand HILL 1984; DIMPFLand ECHOLS1989). The results of the segregation analysis are presented The duplication assay relies on the glySL cold-sensitive mu- tant allele of the glycyl-tRNA synthetase gene, in conjunc- in Table 3. Among 1 1 large colonies tested for each tion with trpA36glyU(sup). The trpA36 mutation is effectively strain, the mutL, mutS and mutH strains were found suppressed by the glyU(sup) allele at 37". However, at 20" to give 100% with unstable phenotype. In addition, suppression of trpA36 is inefficient because ofpoor charging the percentage of segregation was indistinguishable of the suppressor tRNA, leading to poor growth in the between the wild type and the mutL or mutS deriva- absence of tryptophan. In a glySL duplication, the glycine- inserting missense suppressors are charged more efficiently tives. This observation indicates that the frequency of because of a gene dosage effect, leading to better growth homologous recombination between duplicated re- and large colony formation in the absence of tryptophan. gions responsible for segregation was approximately Southern hybridization has shown that this selection is spe- the same for the three strains. Thus the enhanced cific for duplication of the rhsA-rhsB region, which includes duplication formation in the mismatch-defective mu- the glySL gene (LIN, CAPACEand HILL 1984). Duplication frequency was deduced from the ratioof large colonies over tants appears to bespecific for mismatched DNA total colonies (20 plates scored per experiment). Asmis- sequences (although our experiments do not demon- match-deficient strains are known to favor transposon exci- strate that the same sequences are recombined in the sion (LUNDBLADand KLECKNER1984), several large colonies mismatch mutants and wild-type strains). The uvrD from each experiment were checked forthe antibiotic marker linked with the mismatch mutation; 45 among 45 strain gave 0/11 unstable phenotypes; therefore, du- had retained the antibiotic-resistant phenotype. plication frequency might be overestimated in the Segregation analysis of duplications: These measure- uvrD strain. ments were based on techniques developed for Salmonella From the data of Table 2 and Table 3, we infer (ANDERSON,MILLER and ROTH 1976). To remove selective that the MutL and MutS proteins are likely to have pressure favoring glySL duplications, individual large colonies were grown at 37 " in LB broth for10 to 15 generations, an active role in limiting the frequency of large dupli- and then plated on the original selective medium without cations. The MutL and MutS proteins are involved in tryptophan at 20" for 4 days. If segregation occurred, a the initial recognition of mismatched heteroduplex mixed population of large and smallcolonies could be DNA(GRILLEY et al. 1989; MODRICH 1989). MutS detected, among which the proportion of small colonies binds to the mismatch, and MutL associateswith represented the proportion of segregants. MutS. The MutH protein nicks at an unmodified

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