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Reca-Dependent Mutants in Escherichia Coli Reveal Strategies to Avoid Chromosomal Fragmentation

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Reca-Dependent Mutants in Escherichia Coli Reveal Strategies to Avoid Chromosomal Fragmentation RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation Elena A. Kouzminova, Ella Rotman, Lee Macomber, Jian Zhang, and Andrei Kuzminov* Department of Microbiology, University of Illinois at Urbana–Champaign, Urbana, IL 61801-3709 Edited by Charles M. Radding, Yale University School of Medicine, New Haven, CT, and approved October 4, 2004 (received for review August 12, 2004) RecA- and RecBC-catalyzed repair in eubacteria assembles chromo- mutants are predicted to have elevated levels of chromosomal somes fragmented by double-strand breaks. We propose that recA fragmentation. mutants, being unable to repair fragmented chromosomes, de- There are three knockout mutants in E. coli that cause RecA pend on various strategies designed to avoid chromosomal frag- dependence. The dam mutants are deficient in GATC site- mentation. To identify chromosomal fragmentation-avoidance specific DNA adenine methylase (17). Replication-caused tran- strategies, we screened for Escherichia coli mutants synthetically sient hemimethylation of GATC sites is used by E. coli to identify inhibited in combination with recA inactivation by identifying the old DNA strand as the proper template for mismatch repair, clones unable to lose a plasmid carrying the recA؉ gene. Using this whereas lack of GATC methylation in dam mutants results in screen, we have isolated several RecA-dependent mutants and double-strand DNA breaks due to a disoriented mismatch repair assigned them to three distinct areas of metabolism. The tdk and (18). The rdgB mutants depend on RecA and degrade their Ϫ rdgB mutants affect synthesis of DNA precursors. The fur, ubiE, and chromosomal DNA in recA conditions (19), because they are ubiH mutants are likely to have increased levels of reactive oxygen deficient in ITP͞XTPase, an enzyme believed to intercept the species. The seqA, topA mutants and an insertion in smtA perturb- improper DNA precursor dITP, thus preventing hypoxanthine ing the downstream mukFEB genes affect nucleoid administration. incorporation into DNA (20). In the absence of RdgB, hypo- All isolated mutants show varying degree of SOS induction, indi- xanthine is proposed to occasionally incorporate into DNA in cating elevated levels of chromosomal lesions. As predicted, mu- place of guanine, its subsequent excision by EndoV translating tants in rdgB, seqA, smtA, topA, and fur show increased levels of into chromosomal fragmentation (20). Last, fur mutants are chromosomal fragmentation in recBC mutant conditions. Future deficient in the repression of iron assimilation and, as a result, characterization of these RecA-dependent mutants will define experience iron overload (21), which is postulated to lead to mechanisms of chromosomal fragmentation avoidance. increased oxidative DNA damage and RecA dependence in aerobic conditions (22). It is not known whether fur mutants suffer from chromosomal fragmentation. hromosomal fragmentation due to double-strand DNA The dam, rdgB, and fur mutants identify three potential breaks and disintegrated replication forks is a major con- C strategies to avoid chromosomal fragmentation: (i) guidance of tributor to genome instability in all organisms (1, 2). The two proteins that can damage DNA directly (the way Dam methyl- major pathways used by eukaryotic cells to repair fragmented ation guides mismatch-repair enzymes); (ii) quality control in the chromosomes are nonhomologous end joining and homologous DNA precursor metabolism (rdgB); and (iii) reduction in the recombination (3). Nonhomologous end joining is an imprecise level of DNA-damaging species (fur). To find other strategies of repair, frequently involving loss of genetic information, but it is chromosomal fragmentation avoidance or other activities that nevertheless an efficient way to repair double-strand breaks in use the known strategies, we designed an approach to system- cells of higher eukaryotes (4). However, disintegrated replica- atically search for RecA-dependent mutants in E. coli. We then tion forks, having a single double-strand end, cannot be reas- tested the isolated mutants for elevated levels of chromosomal sembled by nonhomologous end joining and require error-free fragmentation. recombinational repair (5, 6). The importance of recombina- tional repair in higher eukaryotes is illustrated by the inviability Materials and Methods of recombinational repair mutants in mice (7) and by the rapid Media, growth conditions, bacterial strains and plasmids, inser- death of recombinational repair-defective vertebrate cells due to tional mutagenesis, color screen, and mutant sequencing are chromosomal fragmentation (8). In the model eubacterium described in Supporting Text, which is published as supporting Escherichia coli, recombinational repair of fragmented chromo- information on the PNAS web site; see also Tables 1 and 2, which somes is catalyzed by the RecA and RecBCD enzymes (9). are published as supporting information on the PNAS web site. RecBCD is an exonuclease͞helicase that prepares the double- strand DNA ends of the break for RecA polymerization (10). SOS Induction. The level of SOS induction due to a particular RecA filament catalyzes homologous strand exchange of the mutation was measured in a derivative of AK43 (into which the broken DNA duplex with an intact sister duplex, thus creating an mutation was introduced by P1 transduction to kanamycin opportunity for double-strand break repair (11). resistance) according to the protocol of Miller (23) with the In recA or recBC mutants of E. coli, double-strand DNA breaks following modifications: (i) cells were grown in LB at 42°C are not repaired (12) and cause chromosomal loss and cell death without antibiotics; (ii) 200 ␮l of the culture with OD600 between (13, 14). However, recA mutant E. coli strains are still 50% viable 0.5 and 0.8 was combined with 2 ml of Z buffer (100 mM Na (15, 16), which indicates that the chromosomal fragmentation is Phos, pH 7.0͞10 mM KCl͞ 1 mM MgSO4͞50 mM 2-mercapto- not a frequent event in E. coli and suggests the existence of ethanol) containing 0.00125% SDS and 25 ␮l of chloroform; (iii) strategies designed to avoid chromosomal fragmentation. Inac- 60 ␮lof4mg͞ml O-nitrophenyl ␤-D-galactoside in Z buffer was tivation of one of these hypothetical avoidance activities should make the corresponding mutant dependent on recombinational repair (‘‘synthetically inhibited’’ in combination with recA inac- This paper was submitted directly (Track II) to the PNAS office. tivation), because both the avoidance of chromosomal fragmen- Abbreviation: IPTG, isopropyl ␤-D-thiogalactoside. tation and the subsequent recombinational repair are not avail- *To whom correspondence should be addressed. E-mail: [email protected]. able in such a double mutant. Moreover, such RecA-dependent © 2004 by The National Academy of Sciences of the USA 16262–16267 ͉ PNAS ͉ November 16, 2004 ͉ vol. 101 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405943101 Downloaded by guest on September 26, 2021 added to the opened cells; and (iv) color development was for 10 min with shaking at 28°C. Quantification of Chromosomal Fragmentation by Pulsed-Field Gel Electrophoresis. Overnight 2-ml LB cultures were shaken at 22°C. In the morning, the cultures were diluted to OD600 0.02–0.06 (for further growth at 22°C) or to OD600 0.001–0.02 (for further growth at 37°C) into 2 ml of LB medium supplemented with 10 ␮Ci͞ml [32P]orthophosphoric acid (1 Ci ϭ 37 GBq). Variations in dilutions accommodate slower growth of some strains. All strains were grown at 22°C for 1 h, and then 37°C samples were shifted to 37°C for 4 h. The 22°C samples were left at 22°C for another5hofgrowth.Growing cultures (OD600 for 37°C samples varied from 0.7 to 1.1; for 22°C samples, variation was from 0.4 to 0.6) were normalized to OD600 0.35, and 0.5 ml of the normalized cultures was pelleted and washed once with 1 ml of TE buffer (10 mM Tris͞1 mM EDTA, pH 8.0) to remove unincorporated radioactivity. Plug preparation was as described (20). Half of the plug was inserted in 1% agarose gel on 0.5 ϫ TBE buffer (89 mM Tris͞89 mM boric acid͞2.0 mM EDTA, pH 7.8) and run for 24 h at 6 V͞cm with a switch time ramp 60–120 seconds, in CHEF-DRII Pulsed-Field Gel Electrophoresis (Bio- Rad). The portion of the gel containing the Yeast Chromosome PFG Marker (New England Biolabs) was stained in 1 ␮g͞ml ethidium bromide solution to estimate the size of fragmented chromosomes. The portion of the gel with radioactive samples was vacuum-dried for2hat80°C to 3MM Whatman paper and directly scanned by PhosphorImager (FujiFilm FLA-3000, Fuji). The fraction of chromosomal DNA escaping from the well into Fig. 1. The color screen for recA-dependent mutants. The screen looks for the inability to lose a temperature-sensitive plasmid carrying the recAϩ gene. the gel was calculated by dividing the counts found in the gel ϩ ϩ (starting at a position halfway between the center of the well and (A) The properties of the strain S295 [lacZ recA p(ori-Ts)-lacZ -recA ] at three temperatures, plated on MacConkey-lactose agar. S295 is lacZϩ recAϩ at 28°C, the center of the compression zone) by the total amount found ϩ ϩ lacZ recA mutant at 42°C, and a mixture of lacZ recA and lacZ recA mutant in the whole lane, including the well. cells (sectoring colonies) at 34°C. (B) The scheme of the experimental strain and the expected colony phenotype of a recA-dependent mutant (converging GENETICS Results arrows). (Inset) Test plating of the original S295 strain and its two derivatives Color Screen. To search for chromosomal fragmentation avoid- carrying mutations known to confer recA dependence, S296 (S295 rdgB3) and ance activities, we set up a screen for recombination-dependent S297 (S295 dut-1), at 34°C. Sectoring colonies are those of the original strain, mutants in E. coli consisting of a lacZ recA mutant strain, which the small nonsectoring colonies are dut-1 mutants, and the midsize nonsec- harbors a lacZϩ recAϩ plasmid with a temperature-sensitive toring colonies are rdgB3 mutants.
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