Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2281-2285, April 1989 Genetics Repair of the chromosome after in vivo scission by the EcoRI (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 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 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. TS6 Arg-56--Gln - + +++ ++++ TS9 Met-255--le + ++++ ++++ +++++ DNA sequencing revealed that a C -* T change alters amino acid 261 from threonine to isoleucine. pJH12 TS0AEcoRI site - - + +++ pJH125 TS6A&EcoRI site - + +++ ++++ By a slight modification, additional TS alleles were iso- lated. A derivative of the endonuclease plasmid pJH10 pJH71 AEcoRI endo +++++ +++++ +++++ +++++ (pJHl5b) carrying the fl intergenic region (packaging signal TS#/pJC1 +++++ +++++ +++++ +++++ and origin of replication) was packaged into transducing Colony growth describes colony size, morphology, and efficiency particles by fl helper phage infection. After infection with of plating (EOP). Scoring is as follows: + + + + +, Large, thick, and round colonies; EOP =0.5-1. ++++, Medium- to normal-sized pJHl5b transducing particles, most of the surviving trans- colonies with ruffled edges; EOP= 0.1-0.5. +++, Medium-sized duced colonies carried EcoRI endonuclease mutants. By this colonies that are thin and mottled; EOP = 0.01-0.1. ++, Small means, the TS3 (Leu-263 -* Phe), TS6 (Arg-56 -* Gln), and colonies that are very flat and mottled; EOP = 0.001-0.01. +, Tiny TS9 (Met-255 -- Ile) mutants were isolated as spontaneous colonies that are very flat and mottled; EOP = 0.001-0.01. -, No mutants. viable colonies; EOP < 0.0001. Downloaded by guest on September 25, 2021 Genetics: Heitman et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2283

Table 3. Restriction of A vir by EcoRI TS mutations induce the SOS response (15). SOS induction can be mea- Mutant 300C 340C 370C 420C sured with strains that carry SOS-inducible loci fused to the wt 1 x 10-4 4 x 10-5 4 x 10-5 4 x lac operon (14). After DNA damage, these strains produce 10-5 f3-galactosidase, which can be assayed by colony color TSO 7 x 10 2 x 10-4 2 x 10-4 2 x 10-2 intensity on X-Gal medium or by cleavage of the chromoge- TS3 1 1 1 1 nic TS6 3 x 104 3 x 10-4 6 x 10-3 0.8 substrate o-nitrophenyl 83-galactoside. TS9 3 x 1o-4 x To measure SOS induction by EcoRI scission, plasmid ND 7 10-2 0.5 pJH10(TS0) was introduced into strain JH39, which carries None 0.8 1.0 1.0 0.9 the lac operon fused to the DNA damage-inducible locus A plating efficiency was measured on strain K91(pJC1) carrying dinD (14). Fig. 3 shows that when strain JH39(TSO) was these EcoRI mutations. Cells were grown at the indicated temper- shifted from 42°C to 37°C, f8-galactosidase expression in- atures in A broth supplemented with maltose, infected with A phage, creased 6- to 8-fold. This plated, and incubated overnight. Values represent the ratio of the induction was blocked by plasmid phage titer on strain K91(pJC1) bearing the indicated EcoRI allele pJC1, which expresses the EcoRI methylase, because strain divided by the titer on K91 alone at 370C. wt, K91(pJH10 and pJC1). JH39(TSO and pJC1) formed white colonies on X-Gal indi- None, Strain K91; ND, not determined. cator medium at all temperatures. Temperature shifts of strain JH39 alone had no effect. When strain JH39(TSO) was As shown in Fig. 2, when cells expressing the TSO allele were shifted from 42°C to 30°C, growth ceased rapidly with no shifted from growth at 42°C to 37°C or 30°C, plasmid increase in /3-galactosidase. Apparently under these condi- pJH10(TSO) DNA was degraded (lanes 7 and 8). However, tions the cell is killed before the SOS response is induced. the linear cleavage product was not seen even when DNA Induction by DNA scission was seen with all the TS mutants. was prepared by a nondenaturing procedure (25) or from a A plasmid encoding the wt endonuclease and a TS EcoRI recB- host that lacks exonuclease V (data not shown). The methylase gave similar results. cleavage and subsequent degradation occurred in vivo, be- In control experiments, mitomycin C at 1 ug/ml induced cause plasmid pBR322 DNA added exogenously during the SOS response 20- to 22-fold above background. The plasmid preparation did not suffer degradation (data not greater induction seen with mitomycin C compared with shown). Chromosomal DNA was also degraded; DNA frag- EcoRI scission may result from a difference in the amount or ments larger than the linear plasmid were seen (lanes 7, 8, 10, in the nature of the inducing lesion. and 11). In contrast, a TSO derivative lacking the EcoRI site SOS Induction Is not Required for DNA Scission Repair. We (pJH12) remained intact and supercoiled (lanes 10 and 11), next asked whether repair of DNA scission requires SOS and similarly, expression of the EcoRP methylase from induction. We tested SOS induction after DNA scission by plasmid pJC1 prevented degradation (lanes 4 and 5). Mutants TSO, TS3, TS6, and TS9 EcoRI alleles in dinDl::lacZ fusion TS3, TS6, and TS9 yielded similar results. We conclude that strains carrying mutations known to block SOS induction by DNA scission has occurred in vivo at EcoRI sites, and then some (recB21, strain JH117) or all agents (recA56, strain JH59 the linear DNA molecules are degraded. and lexA3, strain JH154). In contrast to the parent strain The activity of these mutant proteins was also assayed in JH39, which on X-Gal medium yielded blue colonies indic- vitro with cell extracts prepared under conditions where ative of SOS induction at temperatures where DNA scission endonuclease activity should be maximal. Cells expressing occurred, the recAS6, lexA3, and recB21 mutant strains the TSO mutant yielded EcoRI-specific endonuclease produced white colonies and thus do not induce the SOS with activity comparable to cells expressing the wt protein, response after DNA scission. whereas extracts containing the TS3, TS6, or TS9 mutant We also tested the sensitivity of mutant strains to DNA proteins showed decreased activity (5, 10, and 10% activity, cleavage by either the TS6 or TS9 EcoRI allele. An isogenic respectively; data not shown). set of strains that lack any Mu d lysogen was constructed In Vivo DNA Scission Induces the SOS Response. Earlier [K91 (wt), JH27 (recA56), JH20 (lexA3), JH145 (recB21), and findings suggested that DNA scission might, in general, JH144 (recN262)], because the Mu Gam protein can bind to and protect DNA ends (26). Sensitivity to DNA scission was Plasmid: pJUIU! T0O) pJIfI0VLSO pIu10 rso P!ji12 assayed by determining colony-forming efficiency and - pY( Treatment: iIt 12 37 :30 42 37 A1 42 :17 u"c

1 200- 0C1 CD 150- RFI 0

CD 100-

Lane: 1 2 3 -1 5 6 7 8 9 10 11 D 50- S FIG. 2. In vivo DNA scission by the EcoRI endonuclease. Strains :~~~ and JH11[pJH10(TS0) pJC1], JH11[pJH10(TSO)], and JH11(pJH12) 0 1 2 3 4 5 6 were grown at 42°C until OD6w = 0.2, and then the culture was divided and portions were grown at 42°C, 37°C, and 30°C for 3 hr. Time (hours) DNA was prepared by the alkaline lysis method (16). Lanes 1 and 2 are controls showing uncut (U) and EcoRI-cut (R) pJH10(TS0). DNA FIG. 3. Kinetics of /3-galactosidase induction in strain JH39 from 3- to 5-fold more cells was loaded in lanes 7, 8, 10, and 11 [dinDl::Mu dI(Apr lac)] and JH39[pJH10(TSO)]. Cells were grown at (nonpermissive temperatures) compared with lanes 6 and 9 (permis- 420C in K120 minimal medium/0.2% glucose/0.4% Casamino acids sive temperature). The band labeled X often appears during alkaline (15). Cultures were shifted from 42°C to 37°C or mitomycin C was lysis DNA preparation and may be improperly renatured ("col- added to 1 ,g/ml at T = 0.5 hr. Samples (200 Al) were removed, and lapsed") molecules. Plasmids pJH10 and pJH12 have one and no ,8-galactosidase activity was assayed as described by Miller (22). o, EcoRI sites, respectively. RFI, replicative form I (circular, super- JH39[pJH10(TSO)] at 42°C; *, JH39[pJH10(TSO)] at 37°C; o, JH39 at coiled) DNA; RFIII, replicative form III (linear) DNA. 37°C; and *, JH39 plus mitomycin C at 1 ,g/ml. Downloaded by guest on September 25, 2021 2284 Genetics: Heitman et al. Proc. NatL. Acad. Sci. USA 86 (1989) growth at 420C, 370C, 340C, and 300C (as in Table 2). conditions, intracellular substrates with EcoRI recognition Alternatively, colony-forming efficiency was determined at sites suffer cleavage, whereas those substrates lacking sites the permissive temperature (420C) after incubation for in- or bearing modified EcoRI sites are spared. This in vivo DNA creasing time at the nonpermissive temperature (300C) (as scission induces the SOS DNA repair response. Expression described for Fig. 4). Sensitivity was measured over a range of the EcoRI methylase prevents both cell killing and SOS of doses at which 10-4 to >50% of wt cells survive. By both induction by these alleles. Neither cell killing nor SOS methods these mutant strains are no more sensitive to DNA induction requires a plasmid-borne EcoRI site. We conclude scission than the isogenic parent. SOS induction and cell that in vivo DNA scission at EcoRI sites in the chromosome lethality did not require a plasmid-borne EcoRI site, as induces the SOS response and at higher doses kills the cell. derivatives lacking the EcoRI site yielded similar results. The recent finding that TnWO transposition induces A lysogens DNA Ligase Mutants are Sensitive to DNA Scission. These suggests that DNA scission by transposase may act similarly findings suggested that EcoRI scissions are repaired by (27). mechanisms other than SOS and recombination. We there- Because EcoRI scission induces the SOS response, we fore tested strains deficient in DNA ligase activity for wondered whether blocking this induction would increase sensitivity to DNA scission. In this case, the host strains cell sensitivity to DNA cleavage. SOS induction results from carry TS ligase alleles (lig4 and lig ts7) (17, 18), so an EcoRI a cascade of events (6). After DNA damage, the RecBCD or allele with weak activity at all temperatures (RA2, Gly-19 -+ RecF proteins produce an intermediate that activates the Asp) served to deliver DNA breaks. The lig4 strain N1626 RecA protein. Activated RecA then stimulates autodigestion carrying the plasmid pJHl5b(RA2) grew normally at 300C. of LexA protein, the repressor of the SOS genes. Strains However, when incubated for increased time at 420C, where carrying the recA56 or lexA3 alleles cannot cleave the LexA the mutant DNA ligase activity is greatly reduced, the repressor to induce the SOS response. We found that DNA colony-forming efficiency of N1626(RA2) decreased by a scission did not induce the SOS response in recA- and lexA3 factor of 4000 compared with the lig+ strain N2603(RA2), mutant strains. However, the absence of SOS induction did with N1626 bearing the control plasmid pJH71, or with not increase the sensitivity of these strains to DNA scission. N1626(RA2) also carrying the methylase plasmid pJC1 (see Thus, SOS induction is not required to repair DNA cleaved Fig. 4). When we tried to introduce the RA2 allele into a strain by EcoRI. In strains lacking the RecBCD helicase activity, carrying the more severe lig ts7 allele (N2604), the transfor- SOS is induced by some agents (mitomycin C, UV) but not mation efficiency was reduced 1600- to 2400-fold compared others (nalidixic acid) (28). We find that SOS induction by with the control plasmid pJH71 or 1100-fold compared with DNA scission proceeds via the RecBCD pathway, but a transformation of the isogenic lig+ strain N2603 (data not recB21 mutant strain was not more sensitive to DNA scis- shown). EcoRI methylase expression reversed this lethal sion. The RecBCD complex may simply enter at EcoRI effect. Again, similar results were obtained with a derivative breaks, which are then resealed. Although the RecBCD (RA2ARI, pJH124) that lacked the EcoRI site. proteins exhibit potent exo- and endonuclease activity in vitro, under in vivo conditions the RecBCD helicase activity is more predominant (29). Translocation and unwinding by DISCUSSION helicase would produce single-stranded DNA to activate We have isolated a set of TS EcoRI endonuclease alleles. RecA protein. RecBCD could escape by producing a gap or Strains harboring these mutants are viable at 420C but grow nicking at chi sites. In this way, the SOS response could be poorly or die at lower temperatures. Under semipermissive induced by DNA breaks but plays no part in their repair. In addition to their role in SOS induction, the RecA and 0 U RecBCD proteins participate in homologous recombination. The recA and recB mutant strains are not more sensitive to EcoRI scissions, suggesting that these breaks do not require recombination for repair. In marked contrast are the obser- vations that, compared with isogenic recA+-carrying strains, recA- mutant strains are 104-fold more sensitive to y-ray irradiation (7) and 105-fold more sensitive to cleavage by another endonuclease, A terminase (30). Our results are not attributable to different doses of DNA cleavage, because cE * over a range at which 10-4 to >50%6 of recA+ cells survive, 0 ° 10-2 isogenic recA- cells are not more sensitive. Furthermore, a strain lacking the RecN protein, which plays a role in the more C: 103 repair of y-ray-induced DNA breaks (7), was not co sensitive to EcoRI-mediated DNA scission. These results 0 may be explained by the nature of the breaks, because y-ray cleavage releases a base and leaves nonligatable termini (11), L. whereas EcoRI scission produces staggered breaks with IL ligatable termini. In the case of A terminase, the protein remains tightly bound to one end of the DNA after cleavage (31). This lesion may also require recombination for repair. 104 4 8 12L 16L 20 24 Because DNA ligase is involved in most DNA repair processes by rejoining DNA molecules, we expected this Time at 420c (hours) enzyme to play a role here. As described for Fig. 4 and in Results, conditional DNA ligase mutations conferred ex- FIG. 4. Sensitivity oflig4 mutant to DNA scission. Cultures were treme to DNA scission. The lig4 allele decreases grown at 30°C and serially diluted; then 20-,ul portions were spotted sensitivity on solid medium. Colony formation was challenged by prior incu- DNA ligase activity at 30°C (35% ofwt levels) and 42°C (<1% bation for increased time at the nonpermissive temperature (420C), wt levels) with no loss in cell viability (17, 18). The lig ts7 followed by growth at the permissive temperature. The fraction of allele impairs ligase activity more severely (5% of wt at 30°C) surviving colonies was calculated. *, N1626(RA2); *, N2603(RA2); and does not support cell growth at 420C (<1% wt level) (17, o, N1626(RA2 and pJC1); and n, N2603(RA2 and pJC1). 18). An EcoRI endonuclease allele with reduced activity Downloaded by guest on September 25, 2021 Genetics: Heitman et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2285

(RA2) was inviable in a lig ts7 strain and dramatically reduced 4. Krasin, F. & Hutchinson, F. (1977) J. Mol. Biol. 116, 81-98. the colony forming efficiency of a lig4 strain. Thus, the cell 5. Krasin, F. & Hutchinson, F. (1981) Proc. Natl. Acad. Sci. USA is extremely sensitive to DNA scission when ligase activity 78, 3450-3453. 6. Walker, G. C. (1984) Microbiol. Rev. 48, 60-93. is reduced to 5-<1% of wt levels. We conclude that DNA 7. Picksley, S. M., Attfield, P. V. & Lloyd, R. G. (1984) Mol. ligase is required and may be sufficient to repair these double- Gen. Genet. 195, 267-274. strand DNA breaks. These findings suggest that the tertiary 8. Weibezahn, K. F. & Coquerelle, T. (1981) Nucleic Acids Res. or nucleoid structure of the chromosome (32) may hold 9, 3139-3150. severed ends in proximity and promote their ligation. For 9. Friedberg, E. C. (1985) DNA Repair (Freeman, New York). example, the sedimentation properties of isolated E. coli 10. Lydersen, B. K. & Pettijohn, D. E. (1977) Chromosoma 62, 199-215. nucleoids are not altered when up to 50 double-strand breaks 11. Henner, W. D., Rodriguez, L. O., Hecht, S. M. & Haseltine, are delivered by in vitro y irradiation (10). Alternatively, the W. A. (1983) J. Biol. Chem. 258, 711-713. cell may simply heal each break as fast as it is formed, thus 12. Roberts, R. J. (1976) CRC Crit. Rev. Biochem. 4, 123-164. solving the problem of which ends to ligate together. In 13. Hedgpeth, J., Goodman, H. M. & Boyer, H. W. (1972) Proc. addition, some proteins (Mu Gam, RecA) are known to bind Natl. Acad. Sci. USA 69, 3448-3452. DNA ends and could assist DNA ligase (26, 33). 14. Kenyon, C. J. & Walker, G. C. (1980) Proc. Nat!. Acad. Sci. DNA double-strand breaks stimulate recombination in E. USA 77, 2819-2823. 15. Heitman, J. & Model, P. (1987) J. Bacteriol. 169, 3243-3250. coli and in yeast (1, 2). In E. coli, DNA breaks (including 16. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular EcoRI breaks) activate both the A Red pathway and chi- Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold mediated RecBCD recombination (34-36). DNA breaks may Spring Harbor, NY). promote exchange after exonuclease processing to a strand- 17. Gottesman, M. M., Hicks, M. L. & Gellert, M. (1973) J. Mol. invasive substrate that increases recombination at the break, Biol. 77, 531-547. or by allowing recombinases entry to stimulate recombina- 18. Konrad, E. B., Modrich, P. & Lehman, I. R. (1973) J. Mol. tion away from the break (36). Our findings suggest that Biol. 77, 519-529. 19. Newman, A. K., Rubin, R. A., Kim, S.-H., & Modrich, P. EcoRI DNA breaks act as transient entry sites for the (1981) J. Biol. Chem. 256, 2131-2139. RecBCD complex. Because RecBCD requires DNA ends for 20. Shapira, S. K., Chou, J., Richaud, F. V. & Casadaban, M. J. its recombination activity, there may be an EcoRI cellular (1983) Gene 25, 71-82. analog to provide RecBCD entry points. 21. Cheng, S.-C. & Modrich, P. (1983)J. Bacteriol. 154, 1005-1008. Lastly, we have exploited the finding that DNA scission 22. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold induces SOS::IacZ fusions to screen for and assay EcoRI Spring Harbor Lab., Cold Spring Harbor, NY). endonuclease mutants (unpublished results). This SOS in- 23. Russel, M., Kidd, S. & Kelley, M. R. (1986) Gene 45, 333-338. 24. Shortle, D. (1983) Gene 22, 181-189. duction assay may prove useful for studying other restriction- 25. Zinder, N. D. & Boeke, J. D. (1982) Gene 19, 1-10. modification systems. 26. Akroyd, J. & Symonds, N. (1986) Gene 49, 273-282. 27. Roberts, D. & Kleckner, N. (1988) Proc. Natl. Acad. Sci. USA We thank B. Bachman, M. Casadaban, A. J. Clark, N. Davis, P. 85, 6037-6041. Modrich, and G. Walker for providing strains and plasmids; Gordon 28. McPartland, A., Green, L. & Echols, H. (1980) Cell 20, 731- Lindberg for suggestions; Mark Kelley and Tracy Ripmaster for 737. assistance with DNA sequencing; David Thaler and Frank Stahl for 29. Taylor, A. & Smith, G. R. (1980) Cell 22, 447-457. a preprint of their review; Benedicte Michel for generous discus- 30. Murialdo, H. (1988) Mol. Gen. Genet. 213, 42-49. sions, experimental suggestions, and helpful criticism of the manu- 31. Feiss, M. & Kobayashi, W. (1983) Proc. Nat!. Acad. Sci. USA script; and Marjorie Russel for helpful reading of the manuscript. 80, 955-959. This work was supported by grants from the National Science 32. Worcel, A. & Burgi, E. (1972) J. Mol. Biol. 71, 127-147. Foundation and the National Institutes of Health. J.H. is the 33. Register, J. C., III & Griffith, J. (1986) Proc. Natl. Acad. Sci. recipient of a Medical Scientist Training Program Fellowship. USA 83, 624-628. 34. Stahl, M. M., Kobayashi, I., Stahl, F. W. & Huntington, S. K. 1. Thaler, D. S. & Stahl, F. W. (1988) Annu. Rev. 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