Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2281-2285, April 1989 Genetics Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease (temperature-sensitive EcoRI mutants/DNA ligase/SOS response/DNA repair) JOSEPH HEITMAN, NORTON D. ZINDER, AND PETER MODEL The Rockefeller University, New York, N.Y. 10021 Contributed by Norton D. Zinder, November 22, 1988 ABSTRACT We prepared a set of temperature-sensitive Table 1. E. coli strains mutants of the EcoRI endonuclease. Under semipermissive Strain Relevant genotype Source conditions, Escherichia coli strains bearing these alleles form poorly growing colonies in which intracellular substrates are HB101 recAJ3 Ref. 16 cleaved at EcoRI sites and the SOS DNA repair response is JH11 HB101 recAp This study induced. Strains defective in SOS induction (lexA3 mutant) or JH20 K91 IexA3 Ref. 15 SOS induction and recombination (recA56 and recB21 mu- JH27 K91 recA56 This study tants) are not more sensitive to this in vivo DNA scission, JH39 dinDl::Mu dI(Apr lac) Ref. 15 whereas strains deficient in DNA ligase (lig4 and Ug ts7mutants) JH59 JH39 recA56 Ref. 15 are extremely sensitive. We conclude that although DNA JH117 JH39 recB21 thyA::TnJO This study scission induces the SOS response, neither this induction nor JH137 K91 dinDl::Mu dI(Apr lac) This study JH144 K91 recN262 tyrAl6::TnlO This study recombination are required for repair. DNA ligase is necessary JH145 K91 recB2l thyA::TnJO This study and may be sufficient to repair EcoRI-mediated DNA breaks in JH154 JH39 lexA3 maIE::TnJO This study the E. coli chromosome. K38 HfrC (A) Ref. 15 K91 K38 (A-) Ref. 15 DNA double-strand breaks stimulate recombination in Esch- N1626 lig4 Refs. 17 and 18 erichia coli and yeast (1, 2), and recombination is generally N2603 Iig+ Refs. 17 and 18 thought to repair such DNA breaks (1-3). Repair of DNA N2604 lig ts7 Refs. 17 and 18 scissions has been studied previously using y-ray lesions (4); in E. coli, prior induction of the SOS DNA repair response UV sensitivity, and recN mutants were identified by mito- enhances the repair of DNA cleaved by y rays (5). The SOS mycin C sensitivity. Indicator medium contained 5- response is an altered physiological state that arises after bromo-4-chloro-3-indolyl B3-D-galactoside (X-Gal) at 35 DNA damage and is due to the induction ofa set ofgenes, the Ag/ml. products of which slow cell division and repair DNA (6). To DNA Manipulations. Plasmid pAN4, a pBR322 derivative, repair DNA cleaved by y rays (4, 5, 7) two SOS proteins encodes the EcoRI restriction-modification system and am- involved in recombination, RecA and RecN, and multiple picillin resistance (19). The EcoRI methylase gene of pAN4 copies of the genome are required, suggesting that recombi- was inactivated by inserting a BamHI fragment carrying the nation repairs these lesions. Some -ray breaks may also be kanamycin resistance gene (20) into the Bcl I site within repaired by ligation (8). the methylase gene (Fig. 1). Plasmid pJC1 is a pACYC184 A model for the repair of DNA scission cannot be based derivative that encodes the EcoRI methylase and chloram- solely on the repair of -ray lesions, because y rays not only phenicol resistance (21). Plasmid pJH15b carries the fl cleave DNA but also cause nicks and base adducts and intergenic region (nucleotides 5486-5941) inserted as a Cla I produce free radicals that can oxidize proteins (9). In fact, linked Bgl II fragment in the Cla I site of pJH10 (Fig. 1). single-strand nicks are 10- to 20-fold more abundant than Plasmid pJH71 was derived by partial Pvu II cleavage of double-strand breaks after -ray treatment (10). Further- pJH15b, addition of BamHI linkers, treatment with BamHI more, DNA scission by y rays can release a base and leave and Bgi II, and ligation to delete the N-terminal half of the blunt termini with 5'-phosphoryl and 3'-phosphoryl or 3'- endonuclease gene. phosphoglycolate ends that cannot be ligated (11). Mutagenesis, Mapping, and Sequencing. Mutants were In contrast, restriction endonucleases cleave DNA to yield spontaneous or nitrosoguanidine induced (22). After muta- staggered or blunt double-strand breaks with 3'-hydroxyl and genesis, plasmid DNA was prepared after overnight growth 5'-phosphoryl termini (12). We have studied repair of stag- or cells were grown for 1 hr, infected with fl helper phage gered double-strand DNA breaks delivered with the EcoRI R176 or R189 (23), and grown overnight to obtain a trans- endonuclease, which cleaves within the DNA sequence ducing particle lysate. Mutations were mapped by ligating GAATTC (13). mutant and wild-type (wt) restriction fragments and scoring the phenotype of the hybrid or by heteroduplex deletion MATERIALS AND METHODS mapping (24) with a set of known EcoRI deletions. The relevant region (mutants TSO and TS3) or entire gene (mu- Bacterial Strains. Strains for assaying SOS induction carry tants TS6, TS9, and RA2) was sequenced by the dideoxy- the dinDl::Mu dI(Apr lac) fusion (14) and are listed in Table nucleotide chain-termination method. The RA2 mutant was 1 [see also ref. 15]. Phage P1 transductions were as described isolated from nitrosoguanidine-mutagenized pJH15b plasmid (15). Strains carrying recA, recB, or IexA3 were identified by DNA in strain JH137 by screening for an SOS-induced colony that was viable at 30'C and 420C without methylase. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: TS, temperature-sensitive; X-Gal, 5-bromo-4-chloro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3-indolyl 8-D-galactoside; wt, wild-type. 2281 Downloaded by guest on September 25, 2021 2282 Genetics: Heitman et al. Proc. Natl. Acad. Sci. USA 86 (1989) 0/ 6550 pJH 5blb1 12 TC 4-HindIII 140 I'P2M pJH15b~/3 7000 bp Bg/ II 2880 Bw,,H I 34 Hind III 3085 R+ M R M+ FIG. 1. Compatible plasmid system. The EcoRI endonuclease (R) and methylase (M) genes were cloned on the compatible plasmids pJH10 (or pJHl5b) and pJC1. After mutagenesis the methylase plasmid was linearized to prevent transformation or the endonuclease plasmid pJHl5b was selectively packaged with fl helper phage. single EcoRI site of pJH10 or pJHl5b was destroyed in some The effect of these TS endonuclease alleles on colony cases [TSOARI (pJH12), TS6ARI (pJH125), and RA2ARI growth of strain K91 is shown in Table 2. At semipermissive (pJH124)] by treatment with EcoRI, the Klenow fragment of temperatures, the colonies are flat and translucent and grow DNA polymerase I, and T4 DNA ligase. poorly. By microscopic examination, these cells are fila- Cellular Extracts. Cell paste from 100-ml cultures of strain mented. At 30TC, where the endonucleases are most active, K91 (TS#/pJC1) grown at 30°C was resuspended in 1 ml of some TS mutants impair growth (TS3 and TS9), whereas extract buffer (100 mM NaCI/50 mM Tris-HCl, pH 7.4/10 others are lethal (TSO and TS6). Lethality is not attributable mM MgSO4/bovine serum albumin at 1 mg/ml/1 mM phe- to restriction and plasmid loss, because derivatives lacking nylmethylsulfonyl fluoride) and sonicated (6 x 20 sec) on ice. the EcoRI site [TSOARI (pJH12) and TS6ARI (pJH125)] were After centrifugation for 20 min in a microcentrifuge and 3 hr as lethal (see Table 2). at 100,000 x g, the supernatant was mixed 1:1 with glycerol Restriction of Phage A. Strains bearing these EcoRI muta- and stored at -20°C. tions are rescued for growth by the EcoRI methylase, expressed from plasmid pJC1 (Table 2). Thus, the cleavage specificity of the mutants is unaltered, and the endonuclease RESULTS activity can be assessed by measuring restriction of phage A Isolation of EcoRI Temperature-Sensitive (TS) Alleles. The growth. Table 3 shows that restriction by the wt system was endonuclease and its methylase gene were first separated on unaffected by temperature. In contrast, with the exception of the compatible plasmids pJC1 and pJH10 as described and TS3, restriction by the TS mutants increased dramatically shown in Fig. 1. Methylation protects the cellular DNA from with decreasing temperature. EcoRI-modified A vir plated cleavage, and thus the endonuclease plasmid pJH10 (R+M-, with unit efficiency in all cases (data not shown). For most where R = endonuclease activity and M = methylase mutants, substantial restriction activity was present at tem- activity) is lethal to cells lacking the methylase plasmid pJC1 peratures at which the cell would still be viable without (R-M+). A nitrosoguanidine-mutagenized mixture ofplasmid methylase. DNA was prepared, treated with BamHI to linearize pJC1 In Vivo DNA Cleavage. Plasmid pJH10 carries one EcoRI but spare pJH10, and introduced into strain HB101 or K91. site that should be cleaved in cells bearing a TS EcoRI Because linear DNA transforms wt E. coli at low efficiency, endonuclease allele grown at nonpermissive temperatures. this operation separates plasmid pJH10 from pJC1. Cells that acquire a wt copy of pJH10 (R+M-) suffer DNA degradation Table 2. Effect of EcoRI TS mutants on colony growth of and die. Surviving transformants carry mutants with reduced strain K91 endonuclease activity. By selecting survivors at 42°C and Mutant replica-plating to 30°C, we obtained an EcoRI mutant (TSO) Name Description 300C 340C 370C 420C which was temperature sensitive. Strains K91 or HB101 expressing this allele are viable at 42°C, form colonies poorly TS0 Thr-261-Ile - - + + + + at 37°C, and die at 34°C or 30°C. Thus, the TS EcoRI TS3 Leu-263--oPhe ++ ++++ ++++ +++++ endonuclease activity renders cell growth sensitive to cold.