JOURNAL OF BOACTaaowoy, Feb. 1977, p. 934-k7 Copyright 0 1977 American Society for Microbiology

Vol. 129, No. 2 Printed in U.S.A.

Escherichia coli Mutants Deficient in Exonuclease VII JOHN W. CHASE' AND CHARLES C. RICHARDSON* Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115

Received for publication 23 August 1976

Mutants ofEscherichia coli having reduced levels of exonuclease VU activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One ofthese loci, xseA, lies in the interval betweenpurG and purC; it is 93 to 97% co-tranaducible withguaA. The order of the genes in this region ispurG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaAB. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high fiequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehman (1974). The hyper-Rec character of xseA strains is approximately onehalf that of the

polAxl

defective in the 5'

-.

3'

hydrolytic activity of

deoxyribonucleic acid polymerase I. The double mutant, poLAeXl xseA7, is twice as hyper-Rec as the poU exI mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and Xred grow normally in xseA- strains. A varety of deoxyriboexonucleases are pres- could function in DNA replication, recombinaent in Escherichia coli (Table 1; Table I in tion, and repair (9, 10). Now that mutants havreference 9). Studies with the purified enzymes ing reduced levels of exonuclease VII activity have provided important information concern- have been obtained and one class of them has ing their specificities and usefuilness as re- been mapped, we have initiated studies to deagents in the study of nucleic acids. However, termine if DNA metabolism is affected in muthese in vitro studies have not elucidated the in tants deficient in exonuclease VII. The results vivo role ofthe exonucleases. Therefore, a num- described here show that strains having reber of workers have isolated and characterized duced levels of exonuclease VII activity show mutants of E. coli having reduced levels of increased fiequencies of recombination (hyperRec phenotype), as determined by the procespecific exonucleases (Table 1). We have previously described the isolation dure described by Konrad and Lehman (27) for and characterization of exonuclease VI (9, 10). other mutants ofE. coli defective in DNA repliExonuelease VII is a single-strand-specific exo- cation. We have also found that strains defecnuclease capable of hydrolyzing a deoxyribonu- tive in exonuclease VII activity are more sensicleic acid (DNA) molecule in either the 5' -- 3' tive to nalidixic acid, an inhibitor of DNA replior 3' -- 5' direction. It acts processively, yield- cation (13, 21), and are slightly more sensitive ing oligonucleotide products having 5'-phos- to ultraviolet irradiation than are their parent phoryl and 3'-hydroxyl termini. In an attempt strains. These studies have been facilitated to determine the in vivo function(s) of this nu- greatly by knowledge of the map position of clease, we have isolated mutants having re- xseA, the structural gene for exonuclease VII, duced levels of the enzyme and have initiated in that it has been possible not only to use biochemical and genetic analyses of these isogenic strains, but also to construct strains strains. The symbol xse will be used to refer to carrying specific mutations in conjunction with loci in which mutations occur that affect exonu- the xseA mutation. clease VII activity. MATERIALS AND METHODS The in vitro properties of exQnuclease VII Bacterial and phage strains. All strains used suggest numerous ways in which the enzyme were derived from E. coli K-12. A list of the strains I Present address: Department of Molecular Biology, Al- used and their properties is given in Table 2. Figbert Einstein College of Medicine, Bronx, NY 10461.

ure 1 is a map oftheE. coli chromosome showing the 934

TABLE 1. E. coli deoxyriboexonucleases Deoxyribonuclease

Exonuclease I

Gene"

Map locationb

xonA

44 (33)

sbcB

44 (55)

II III

xthA

38 (44)

IV V

recB

60 (16, 61)

recC

60 (16, 61)

VII

xseA

53 (this paper)

VIII

recE

Types of mutants, phenotype, and commentsf

Other referencesd

Ts (64), deletion (54), reduced activity; mutants partially or fully suppress recB and recC mutations (33, 35, 55) See footnote e Ts (43), reduced activity; mutants simultaneously affect endonuclease II (65) Ts (31, 56), reduced activity; mutants UV, X-ray, mitomycin C sensitive and recombination deficient (16, 61) See footnote e

32, 38, 42

VI

Ts (this paper), reduced activity; mutants hyper-Rec (this paper) Structural gene for an exonuclease that occurs in sbcA mutants resulting in suppression of the recB and recC mutations

(4, 34) DNA polymerase I

Ts (46), amber (14), reduced ac- 20, 24, 36, 57 tivity; polAex hyper-Rec (27) II 2 (7, 25) polB Reduced activity; mutants not 8, 19, 29, 30 characterized with respect to exonuclease activities III polC 4 (59) Ts (18); mutants temperature 39 sensitive for DNA synthesis (59, 60); mutants not characterized with respect to exonuclease activities a See Fig. 1 for the location of these genes on the E. coli genetic map. bNumbers in parentheses refer to mapping references. c Numbers in parentheses refer to references. Ts indicates that the enzymatic activity isolated from the mutant is temperature sensitive. "Reduced activity" refers to mutants having reduced levels of the activity (not temperature sensitive); specific references are not given for these mutants since they can be found among the other references. d See Table I in reference 9 for additional references; those given here are intended to update and correct omissions in those given previously (9). e As we suggested (9) (and as was previously suggested by Wright et al. [62]), the former designations polAe

85 (22)

"exonuclease II" and "exonuclease VI" for the 3'

5' and 5'

3'

hydrolytic

activities associated with DNA

polymerase I, respectively, will no longer be used since these activities are physically part of the polymerase I molecule. All of the original polA mutants isolated on the basis of having reduced levels of the polymerase 3' activity that have been carefully studied (polAl, polA5, and polA12) have nearly normal levels of the 5' hydrolytic activity of the enzyme (36). These mutants have not been assayed specifically for the 3' 5' activity of the

enzyme.

No mutants have yet been described

having reduced

levels of

only the

3'

5'

hydrolytic activity. The polAl07 mutant deficient in the 5' 3' activity of polymerase I retains the 3' 5' activity and the polymerase activity (20). Other mutants deficient in the 5' -+ 3' activity have also been isolated and designated polAex (27), even though this activity is a polA gene product. An obvious problem with the genetic nomenclature of mutations in the exonuclease activities associated with DNA polymerases has thus resulted. The problem will no doubt become even more confusing as defects in the exonuclease activities associated with polymerases II and III are described. We make no suggestions to solve this problem, but merely wish to point out that, if strict genetic nomenclature is to be followed (i.e., the system suggested by Demerec et al. [151), mutations in any activity associated with a particular gene product should contain the designation assigned that gene and an allele number to designate the particular mutation. In this way Glickman et al. (20) assigned poLU107 to the mutation they isolated in the 5'-hydrolytic activity of DNA polymerase I. If this established system is not to be followed, then a new or modified system, possibly one applying only to these special cases, should be considered. But it is most important that it be carefully conceived, unambiguous, and broad enough to include all activities associated with DNA polymerases. 935

936

J. BACTERIOL.

CHASE AND RICHARDSON

TABLE 2. Bacterial strains Genotype

Strain

Source of construction

HfrH

'. thi-i rel-1 A' 4 pil pyrB

E. coli Genetic Stock Center strain 259

Hfr KL16

-i

' thi-i rel-1 XserA lysA

E. coli Genetic Stock Center strain 4245

Hfr KL983

supN dsdA xyl-7 lacYl or lacZ4 mglPi X-

E. coli Genetic Stock Center strain 4240

KL164

KfrKL16, nalB14 thyA24 drm-3 thy-i rel-1

P3478

polAl thyA36 X-

PCO150 PC0631

thi-I his-68 tyrA2 trp-45 purC50 lacYl gal-6 xyl-7 mtl-2 maLA1 strA125 tonA2 tsx-70 A' X- supE44 HfrR4 pyrA35 purG48 metBI rel-i

AT978

HfrKL16 thi-1 rel-1 dapE9 X-

AT2457

HfrH thi-1 glyA6 rel-i A-

AT2465

HfrH guaA21 thi-1 rel-i X

AT2471

HfrH thi-i tyrA4 rel-i X-

KS439b RS5052 PA610

F- lac 080dIIlac metB- ara- thyA- thi F- lac 480dIIlac polAexi ara- thyA- thi F- thr-i leu-6 his-1 argHi lys-25 lacYl malAl xyl-7 ara-

E. coli Genetic strain 4304 E. coli Genetic strain 4303 E. coli Genetic strain 4900 E. coli Genetic strain 4497 E. coli Genetic strain 4544 E. coli Genetic strain 4507 E. coli Genetic strain 4509 E. coli Genetic strain 4510 Konrad (27) Konrad (27) F. Jacob (2)

KLC3 KLC7 KLC8 KLC12 KLC24 KLC26 KLC27 KLC39 KLC46 KLC48 KLC52 KLC54

13 mtl-2 gal-6 purE43 tonA2 thi-i str-9 Ar PA610 xseA3 PA610 xseA7 PA610 xseA8 AT2465 thyA HfrH xseA3 thyA- thi-i rel-i AHfrH xseA7 thyA- thi-i rel-1 XHfrH xseA8 thyA - thi-1 rel-1 XHfrKL16 guaA21 hisF- lac 80dIIlac metB- ara- guaA21 thi F- lac 80dlllac polAexl ara- guaA21 thiF- lac 480dIIlac metB- ara- xseA7 thiF- lac qb8OdIIlac polAexI ara- xseA7 thi

This paper This paper This paper Trimethoprim P1 transduction: KLC3 x KLC12 P1 transduction: KLC7 x KLC12 P1 transduction: KLC8 x KLC12 Chase Hfr cross: KLC39 x KS439b Hfr cross: KLC39 x RS5052 P1 transduction: KLC7 x KLC46 P1 transduction: KLC7 xKLC48

Stock Center

Stock Center Stock Center

Stock Center

Stock Center Stock Center Stock Center Stock Center

positions of relevant markers (3) and the points of prepared as L broth containing 0.6% agar. L broth origin of the Hfr strains used (40). Phage P1- plates containing calcium and chloramphenicol used CMclrlOO was used for generalized transductions in transduction studies were prepared as described (49). Phage T7 was prepared by the method of Stu- by Miller (45). L broth plates containing Giemsa dier (52). Phage fd was prepared according to Miller stain used for fd plating were prepared using L broth (45). Phage Xr,d was prepared from a lysogen agar with the addition of 0.8% Giemsa stain before autoclaving. Lactose tetrazolium indicator plates (CsOOXcI857red3) according to Miller (45). Media. The minimal medium used was that de- were prepared using antibiotic medium no. 2 and scribed by Vogel and Bonner (58) supplemented with contained 1% lactose as described by Miller (45). Mating and transduction procedures. Trwnsduc0.5% of the required sugar, 40 ,ug of any required amino acid per ml, 0.5 ,ug of vitamin Bi per ml, and tions were carried out using PlCMclrlOO as de20 ,ug of any required purine or pyrimidine per ml scribed by Miller (45). In all cases transductants except thymine, which was present at 50 ,ug/ml. The were examined for lysogeny by sensitivity to chlorsame medium was used for minimal plates, but also amphenicol and phage T7. All bacterial matings were performed as decontained 1.5% agar (Difco). L broth was prepared according to Lennox (37) with 50 ,ug of thymine per scribed by Miller (45). Matings were interrupted by ml. L broth plates were prepared from the same 40-fold dilution in 0.85% NaCl and mechanical shakmedium containing 1.5% agar. L broth soft agar was ing, using the apparatus described by Miller (45).

EXONUCLEASE VII MUTANTS OF E. COLI

VOL. 129, 1977

polB

O/C

ooA \ b

po/A 90

>C

V

20-1

980

470 O \

30

04

-

0

40

rec "

,'

L .-.02-

4

1.02-

~~shcA xonA

>

M/NUrES

FIG. 1. Genetic map of E. coli K-12 (after Bachmann et al. [3D showing the location of some of the markers referred to in the text and Table 1 and approximate origin and direction of transfer of Hfr strains used in the study. The expanded portion around min 53 shows the relative distances (in minutes) of xseA from several markers calculated from co-transduction frequencies (see Table 4 and text).

Isolation of mutants. Since it was not possible to develop a selective screening procedure, a mass screening technique such as that used in the isolation of strains having reduced levels of DNA polymerase I (14), DNA polymerase II (8), exonuclease I (65), and exonuclease III (43) was followed. The procedure was similar to that of Milcarek and Weiss (43). E. coli PA610 was mutagenized withN-methylN'-nitro-N-nitrosoguanidine, 1 mg/ml, according to the method of Adelberg et al. (1) in tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (pH 6.1) for 30 min at 30°C to the extent that 3.6% of the cells became rha- and 29% developed auxotropic requirements in addition to those of the parent strain. Preparation of cell extracts. In the semiautomated screening procedure, cell extracts were prepared essentially according to the method of Milcarek and Weiss (43). When extracts were made from a small number of large cultures, cells from a portion of each culture were collected by centrifugation at 10,000 rpm for 10 min in the Sorvall SE-12 rotor. The supernatant fluid was discarded, and approximately 0.25 ml of the lysozyme-Tris-ethylenediaminetetraacetate (EDTA) solution described by Milcarek and Weiss

937

(43) was added for each 2 ml of original cell culture at optical density at 590 nm. The cells were then lysed as described above. When exonuclease VII activity was to be determined quantitatively, extracts were prepared from large cultures by sonic irradiation. Cells were collected by centrifugation at 10,000 rpm in the Sorvall SS-34 rotor and resuspended in 20 mM Tris-hydrochloride buffer (pH 8.0), 10 mM 2-mercaptoethanol, 0.05 mM EDTA, and 20% (wt/vol) glycerol at approximately 0.5 mg of protein per ml. The cells were then disrupted by sonic irradiation for 3 min (six 30s treatments, allowing cooling in between) (Branson Sonifier, model S75, microprobe, power setting 2 A). Assay of exonuclease VII in cell extracts. Exonuclease VII can be assayed specifically in crude cell extracts since it is the only E. coli exonuclease that is fully active in the absence of added divalent cations. We have also made use of a substrate containing 5' single-stranded termini extending from a duplex region (9). The 5' termini ofthese molecules can be labeled with [y-32P]adenosine 5'-triphosphate of high specific activity in the reaction catalyzed by polynucleotide kinase. Since exonuclease VII hydrolyzes only the single-stranded termini of this substrate, the assay is highly sensitive and specific. No E. coli nuclease is known that can interfere with the specificity of the exonuclease VII assay described here. Reaction mixtures contained 67 mM potassium phosphate buffer (pH 7.9), 8.3 mM EDTA, 10 mM 2mercaptoethanol, and 0.5 to 1 nmol of exonuclease Ill-treated [3H,5'-32P]DNA. This substrate contains 5' single-stranded regions extending from a duplex region. The 5' termini of the single-stranded regions are labeled with 32p. Exonuclease VII specifically hydrolyzes only the single-stranded regions, leaving the duplex region intact. The substrate is also uniformly labeled with tritium so that the integrity of the entire molecule can be monitored during the assay. The preparation and use of this substrate have been previously described in detail (9). To assay extracts made in 96-well plates, 50 ,ul of the reaction mixture was added to each well, and the plates were incubated by floating them on a water bath at the desired temperature for 30 min. In the original mutant selection all assays were carried out at 43°C so that temperature-sensitive enzyme activities could be detected. The reaction was stopped by the addition of 25 ,lM of 2.5 mg of salmon sperm DNA per ml and 25 ,ul of 50% trichloroacetic acid. The precipitate was collected by centrifugation of the plate as described above. The radioactivity in a portion of the supernatant fluid was either determined directly by counting in Bray's fluor (6) in a Packard scintillation spectrometer or by spotting on Norit impregnated paper (Schleicher and Schuell, grade 508) and exposing the paper to X-ray film (Kodak, No-Screen, NS54T). Since the supernatant fluid solution is acidic, the 32P-labeled nucleotides absorb strongly to the charcoal, so that a small exposed area is observed on the X-ray film wherever exonuclease VII activity is present. This assay can be made extremely sensitive, reproducible, and semiquantitative. For example, we

938

J. BACTERIOL.

CHASE AND RICHARDSON

have identified strains containing a temperaturesensitive exonuclease VII activity and a reduced level of activity (20% of wild type), as well as those containing essentially no detectable activity (see Results). When individual extracts were prepared from large cell cultures as described above, portions were added directly to a reaction mixture (final volume, 0.15 ml) and assayed as described previously (9). Purification of exonuclease VII. Exonuclease VII was purified from strains PA610 and KLC3 through the diethylaminoethyl-cellulose step as previously described (9). The purified enzyme was assayed by the standard exonuclease VII assay (9). Assay of exonuclease VII in cells obtained from matings and transductions. In all matings and transductions it was necessary to directly assay cells for exonuclease VII activity, since there is no means yet available to select for strains having reduced levels of this activity. For this purpose, mass assays were performed by procedures similar to those used for the original mutant isolation. Individual colonies resulting from matings or transductions were grown in 96-well plates and assayed for exonuclease VII activity as described above. Recombinant cells or those resulting from transductions were normally not repurified. Individual colonies were picked and transferred to 96-well plates containing defined media lacking the particular supplement that the cells should no longer require. No problem was every encountered due to partial growth of either donors or recipients. Even if occasional problems were to occur, it would not be significant, as hundreds of recombinants or transductants were normally assayed in each experiment.

UV light survival curves. Sensitivity to ultraviolet (UV) irradiation was determined as described by Willetts and Mount (61). Cells were grown at 30°C in L broth and plated on L broth plates after UV irradiation. Irradiation was with a General Electric germicidal lamp. The UV dose was 10.2 ergs/mm2 per s, as determined with an International light germicidal radiometer (model IL 570). Protein determination. Protein was determined by the method of Lowry et al. (41). Chemicals. N-methyl-N'-nitro-N-nitrosoguanidine was purchased from Aldrich Chemical Co. Chloramphenicol and nalidixic acid were obtained from Calbiochem.

RESULTS

Isolation of mutants. Approximately 5,000 extracts derived from a stock of cells (E. coli PA610) heavily mutagenized with N-methylN'-nitro-N-nitrosoguanidine were assayed. The screening procedure was designed so that the temperature-sensitive, conditionally lethal mutants could be isolated if the enzyme was essential (see above). Nine strains having reduced levels of exonuclease VII activity were found. These strains were numbered KLC1 through KLC9. Five of these strains (see Table 3) are

TABLE

3. Exonuclease VII activity in

extracts of xse

mutantsa

Strainb Sri

xse

geno-

type

Sp act (pmol/mg of protein)

430C 97.1

PA610

xse+

300C 74.7

KLC1 KLC2

xse-1 xse-2

sls1 1.2 s1

KLC3 KLC4 KLC5

xse-3 xse-4 xse-5

13.6 1.6 S1

KLC6 KLC7

xse-6 xse-7

S1 S1

KLC8

xse-8

1

1

KLC9

xse-9

16.6

18.8

9.6 s1

Q43 3

1.30

0.70

1

1.13

All extracts were prepared by sonic irradiation (see Materials and Methods) from cells grown exponentially at 30°C, and each contained approximately 0.5 mg of protein per ml. The standard exonuclease VII assay (9) was used with the exonuclease IIItreated T7 DNA substrate (see Materials and Methods). Specific activity is given as picomoles of acidsoluble 32p produced in 30 min per milligram of protein. b The groups of strains between lines are known to be of independent origin (see text). c The assay was not sensitive enough for specific activities below 1 to be reliably measured. a

known to be of independent origin, since they were derived from separate cultures from the original segregation flasks (see above). Strains having reduced levels of exonuclease VII activity have been designated xse-. All strains having reduced levels of exonuclease VII activity were examined for growth at 30 and 420C. Only strains KLC1 and KLC2 were found to have obvious reduced survival at 420C. However,

subsequent isolation of temperature-resistant revertants and analysis of their exonuclease

VII activity suggest that this defect is not associated with the reduced level of exonuclease VII activity in these strains. Level of exonuclease VII activity in xse strains. Extracts were prepared from exponentially growing cultures of KLC1 through KLC9 by sonic irradiation (see above). The specific activity of exonuclease VII in each of these strains was determined at 30 and 430C and compared to that of the parent strain (PA610). The results (Table 3) show that seven of the strains isolated (four of which are of independent origin) have little or no detectable exonuclease VII activity at either temperature. One

VOL. 129, 1977

EXONUCLEASE VII MUTANTS OF E. COLI

939

strain (KCL3) contains a temperature-sensitive interval between the points of origin of Hfr exonuclease VII activity and will be considered KL16 and Hfr KL983 (Fig. 1). Further studies in more detail later. The remaining strain by interrupted mating with Hfr KL16 selecting (KLC9) contains 20% of the normal level of for either Lys+ or His+ recombinants placed the exonuclease VII activity at both temperatures. position of the mutation near the point of origin Loss of exonuclease VII activity in mutants of E. coli Hfr KL983 (data not shown). is not due to inhibition of the activity. ExMapping by P1 transduction. Since the letracts were prepared from KLC1, KLC5, KLC7, sion in strain KLC7 appeared to lie in a small and KLC8 and the parent strain (PA610) by region to the left of the point of origin of Hfr sonic irradiation (see above). The activity of KL983 (Fig. 1), co-transduction frequencies exonuclease VII in the parent strain was deter- were determined with several markers in this mined with and without the addition of mutant region. Transduction was mediated by phage extract. The results showed that the addition of PlCMclrlOO. The donor was KLC7 xse-. Selecmutant extract from any of the strains tested tion was for the auxotropic marker indicated did not significantly affect the level ofexonucle- (Table 4). Transductants were assayed for exoase VII activity in the extract ofE. coli PA610. nuclease VII activity is described (see above). This result suggests that the loss of exonuclease The results of these studies (Table 4) produced VII activity in these strains is due to either the linkage map shown in Fig. 1 and indicate reduced amounts of the enzyme or alterations that the xse-7 locus is 94% co-transducible with in the enzyme, and not to the presence of an guaA (linkage, 0.04 min). Therefore, xse-7 lies inhibitor. betweenpurG and purC, probably within a few Initial mapping studies. One strain (KLC7), genes of the gua operon (47, 48). It is not possiwhich grew normally at both 30 and 420C and ble, however, to determine whether the gene had little or no detectable exonuclease VII ac- lies to the left or right ofguaA from these data. tivity (see Table 3), was selected for the initial xse-1, xse-2, xse-4, and xse-5 do not comapping studies. Hfr mating experiments were transduce with guaA. The co-transduction frecarried out to determine the approximate posi- quencies of strains KLC1 through KLC9 were tion of the defect in this strain on the E. coli determined with guaA. Phage PlCMclrlOO was chromosome. Recombinant cells were selected grown on each donor (KLC1 through KLC9). and then assayed for exonuclease VII activity The recipient was E. coli AT2465 guaA21, and using the screening assay procedure (see selection was for Gua+. Transductants were asabove). Approximately 100 recombinants were sayed for exonuclease VII activity by the assayed in each experiment. E. coli Hfr KL16 screening procedure (see above). The results (see Fig. 1 and Table 2) was mated with strain (Table 5) show that not all of the defects resultKLC7 xse-7 his-, and His+ recombinants were ing in reduced levels of exonuclease VII activity grown and assayed for exonuclease VII activity. in these strains are co-transducible with guaA. After a 40-min mating, exonuclease VII activity The defects in five of the original mutants (four was found to appear in recombinant cells. A of which are of independent origin) are 93 to second mating experiment was performed with 97% co-transducible with guaA and are all strain Hfr KL983 (see Fig. 1 and Table 2). Se- probably within the same gene as xse-7 (see lection was again for His+ recombinants, but no Discussion). This locus has been designated exonuclease VII activity was found in any of xseA. these recombinants. These results fixed the poThe mutations causing reduced levels of exosition of the mutation in strain KLC7 in the nuclease VII activity in four of the original TABLE 4. Transduction of the recipient strain to Xse- by Pl phage grown on strain KLC7 Co-transduction Interlocus distance" (mn) frequency (%) 0 >2 0 AT2471 tyrA + 8.7 1.11 23 AT2457 glyA+ 0.2 73 185 PCO631 purG+ 94 0.04 165 AT2465 guaA+ 14.0 39 0.96 purC+ PCO150 28 11.8 1.02 AT978 dapE+ = aThe recipient strain was routinely grown to optical density at 590 nm 0.5. This is equivalent to approximately 2 x 108 cells/ml. In these transductions 2.5 ml of cells was always used. The multiplicity of infection was 2.5. b Determined by Wu's equation (63). Recipient strai. Recipient strain"

Selected marker marker

No. assayed for Xe Xse 256 265 253 176 278 238

No. Xse~

940

J. BACTERIOL.

CHASE AND RICHARDSON

mutants (two of which are of independent ori- at 43°C of exonuclease VII purified from strain gin) do not co-transduce with guaA at any de- KLC3 was fivefold less than that of the enzyme tectable frequency and therefore probably occur purified from strain PA610 (Fig. 3). These reat a locus (or loci) distinct from the xseA locus. sults demonstrate that exonuclease VII in xseA is a structural gene for exonuclease strain KLC3 is temperature sensitive, and VII. The results presented in Table 3 show that therefore xseA is a structural gene for exonuclethe residual exonuclease VII activity present in ase VII. xse strains support the growth of phages T7, extracts of the mutant strain, KLC3, is heat labile. We have purified the enzyme from this fd and k.d. Wild-type T7 phage have the same strain (KLC3) and the parent strain (PA610) efficiency of plating on all xseA strains as on and compared their properties to show that the xseA+ strains. To test the plating efficiency of defect in strain KLC3 is actually in exonuclease phage fd on xseA- strains, the mutations in VII. The ratio of activities at 43 and 30°C (Q30) strains KLC3, KLC7, and KLC8 were transremained constant for the enzyme from both ferred to an F+ strain (AT2465) by P1 transducstrains during the purification, but was approx- tion, producing strains KLC24 F+ xseA3, imately twofold lower with the enzyme purified KLC26 F+ xseA7, and KLC27 F+ xseA8. The efficiency of plating of phage fd on these xseA from strain KLC3 (Table 6). A time course of the exonuclease VII reaction strains was identical to that on the parent at 30 and 43°C using the enzymes purified from strain (AT2465). Phage AXld had normal effithese strains is shown in Fig. 2. At each point ciency of plating on strains carrying the xseA3, during the 30-min reactions, the Q43 of exonu- xseA 7, and xseA8 mutations. xseA strains show slightly increased sensiclease VII activity purified from strain KLC3 tivity to UV irradiation. Exonuclease VII can was twice that of the Q4 of the enzyme purified from strain PA610. Furthermore, the half-life excise thymine dimers from DNA in vitro (10), suggesting a role for exonuclease VII in the repair of UV damage. E. coli xseA mutants TABLE 5. Co-transduction of xse with guaAa are slightly more sensitive to UV irradiation CoNo. of the parent, E. coli KLC12 (Fig. 4). A UVthan traTransduc- xse geno- transduc- No. of strain, E. coli polAexl (27), lacking sensitive xse freXseo astants tional dot 3' hydrolytic activity of DNA polymerthe 5' fresayed for nor quency ase I, is shown for comparison. Xse xseA- strains show increased sensitivity to 0 0 30 xse-1 KLClb nalidixic acid. Strains having reduced levels of 0 0 184 xse-2 KLC2 exonuclease VII were tested for sensitivity to 97 the DNA synthesis inhibitor, nalidixic acid (13, 171 177 xse-3 KLC3 0 0 130 xse-4 KLC4 21), and compared with their parent strain and 0 0 169 xse-5 KLC5 a strain resistant to low levels of nalidixic acid (nalBr) (23). Exponentially growing cells were 96 136 142 xse-6 KLC6 94 plated on L broth plates containing varying 157 167 xse-7 KLC7 concentrations of nalidixic acid and incubated 93 154 165 xse-8 KLC8 at 30°C. The results (Fig. 5) show that a strain containing the xseA7 mutation (KLC26) was 95 159 168 xse-9 KLC9 twice as sensitive to nalidixic acid as the parent aGua+ transductants of strain AT2465 were obtained as strain (KLC12). Strains xseA8 and xseAlO described in the text. showed similar sensitivity. The control nalBr be of bThe groups of strains between lines are known to strain showed the expected sensitivity (resistindependent origin (see text). --

TABLE 6. Purification of exonuclease VII from wild-type and mutant strains KLC3 (xseA3)

PA610 (xse+)

Fraction

Sp acta

Q3

Sp act" 8.59 26.8 223 287

340

0.73 0.76 II. Streptomycin pellet 0.50 III. Pooled diethylaminoethyl 0.56 IV. Concentrated diethylaminoethyl a The specific activity is given for the assay at 300C. The standard exonuclease VII assay was used with the exonuclease III-treated DNA substrate (see text and reference 9). Exonuclease III-treated DNA units are given here in picomoles rather than nanomoles (see reference 9 for definition of units). I. Extract

52.9 302 987 1,528

1.25 1.22 1.24 1.12

VOL. 129, 1977

EXONUCLEASE VII MUTANTS OF E. COLJ

941

PA610 EXO Vn

100

60 40 20 -11

10

KLC3 EXO

6

|~~~~~OA 0.4 _-

4

30°'C

2 0

0.2 -/

,

0 10

_

~

20

~

10



~~~~~30

O

MINUTES FIG. 2. Time course of exonuclease VII reaction using exonuclease VII purified from strains PA610 and KLC3. The enzymes were purified through the diethylaminoethyl-cellulose step of Chase and Richardson (9). A reaction mixture (1.0 ml) was prepared containing 67 mM potassium phosphate buffer (pH 7.85), 8.3 mM EDTA, 10 mM 2-mercaptoethanol, and 6.7 nmol of exonuclease III-treated [3H, 5'32P]DNA (9). The reactions with exonuclease VII derived from strains PA610 and KLC3 contained 0.0086 and 0.0081 exonuclease III-treated DNA units of enzyme, respectively. At the times indicated, 0.15ml samples were withdrawn, and the acid-soluble radioactivity was determined as previously described

20

30

40

M/IVtrES AT

43

50

60

C

FIG. 3. Heat inactivation of exonuclease VII purified from strains PA610 and KLC3. The diethylaminoethyl-cellulose fractions described in Fig. 2 were used. Exonuclease VII derived from strains PA610 and KLC3 were diluted, respectively, to 0.089 and 0.081 exonuclease III-treated DNA units per ml in 50 mM Tris-hydrochloride buffer (pH 8.0), 10 mM 2-mercaptoethanol, and 0.5 mg of bovine serum albumin per ml and incubated at 43°C. At the times indicated, 0.01-ml samples were assayed according to standard assay conditions with the exonuclease III-treated DNA substrate (9). The percentage of decrease in activity is relative to the zero-time sample set at

100.

WILD

10

\>

T

Y

P

TYPE

E

(9).

ant to 4 jig of nalidixic acid per ml but sensitive to 10 ,ug/ml) (23).

Recombination after conjugation occurs efficiently in xseA- strains. We have suggested (10), based on the in vitro properties of exonuclease VII, that the enzyme may serve a role in recombination in E. coli. Conjugational recombination frequencies were determined by mating strains KLC7 xseA 7 and PA610 xseA + with HfrH and Hfr KLC16 strains. The mating with E. coli Hfr H was inter!upted before the xseA + locus could be transferred (Thr+ recombinants selected), whereas the mating with E. coli Hrf KL16 was continued for a period sufficient for the xseA + locus to be transferred (His+ recombinants selected). Recombinants derived from these matings were assayed for exonuclease VII activity (Table 7) to be certain that the xseA + locus was transferred in the mating with KL16 but not in the mating with E. coli HfrH. The results (Table 7) show no defect in recombina-

xseA?

Qz~~

0

~~~~~oAx

100

200

300 400 500 600

UV DOSE (ergs/mm2) FIG. 4. UV irradiation survival curves for xseAAand xseA+ strains. Symbols: El, KLC12 (xseA+); A\, KLC26 (xseA-); 0, RS5052 (polAeWl).

942

i

J. BACTERIOL.

CHASE AND RICHARDSON

60

ii-40

p42.

""20

0.4

0.6

2

NALADIXIC ACID CONCENTRATrION (g/m/) FIG. 5. Comparison of the sensitivity of various strains to nalidixic acid. L broth plates (see text) containing various concentrations ofnalidixic acid were prepared. Exponentially growing cells were diluted in 0.85% NaCl and plated. Plates were incubated for 2 days at 30°C. Colony-forming ability is expressed relative to that on plates containing no nalidixic acid. TABLE 7. Efficiency of conjugational recombinationa Donor (viable cells/ml)

Recipient selection

No. of recombinants/ml

% Recombinants that are Xse+

HfrH (2.8 x 107)

PA610 xseA+ Thr+ Strr 3.2 x 105 KLC7xseA7 2.7 x 105 0 Hfr KL16 (1.1. x 107) PA610xseA+ His+ Strr 1.7 x 104 KLC7 xseA7 2.5 x 104 20 a Matings were carried out in L broth at 37°C (see text). The mating with E. coli HfrH was for 20 min; the mating with E. coli Hfr KL16 was for 40 min.

tion ability in xseA - strains compared to wild razolium indicator plates, these cells initially type (PA610), and thus no dependence of trans- are unable to ferment lactose and form red fer of the xseA + locus for genetic recombination colonies. After prolonged incubation (3 days at in the recipient. 30°C), white lac+ papillae appear on the surface xseA - strains show increased frequencies of of these red colonies. Each of these papillae recombination. Strains having reduced levels represents a clone of lac+ cells resulting from of the 5' -- 3' hydrolytic activity of DNA polym- intrachromosomal recombination between the erase I (polAex-) (See Table 1) have been shown different lac- mutations. Strains that produce to be "hyper-Rec" by Konrad and Lehman (27). an increased number of these white lac+ paSince exonuclease VII is another specific 5' -+ pillae are termed hyper-Rec. In Fig. 6a, strain 3' exonuclease activity that could be involved in KS439b is plated on a lactose tetrazolium indirecombination, it was of interest to determine cator plate showing the "background" level of whether or not xseA - strains also showed the papillae that is always observed. When the hyper-Rec phenotype. polAexl mutation occurs in this strain Konrad and Lehman (27) have constructed a (RS5052), it becomes hyper-Rec, as shown by strain of E. coli (KS439b) containing a lac- the increased number of papillae on the surface mutation at the lac region of the chromosome of the colonies (Fig. 6b). and a different lac- mutation carried on a We constructed an xseA 7 derivative of strain 080dlac prophage. When grown on lactose tet- KS439b (KLC52). When plated on lactose tetra-

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EXONUCLEASE VII MUTANTS OF E. COLI

943

FIG. 6. Strains used to determine hyper-Rec phenotype of exonuclease VII mutants plated on lactose tetrazolium indicator plates. The number of white lactose-positive papillae is a measure of the hyper-Rec character of the strains (see text). (a) KS439b lac 080d11lac; (b) RS5052 lac 480ddIlac; (c) KLC52 lac 480d1Ilac xseA7; (d) KLC54 lac 080d1Ilac poL4exi xseA7.

zolium indicator plates, this strain (KLC52) showed an increased number of papillae compared to the parent strain (Fig. 6c). Thus, strains having reduced levels of exonuclease VII show the hyper-Rec phenotype. Similar results have been obtained with the xseA8 mutation. Finally, a double mutant, E. coli KLC54 polAexl xseA7, was prepared. It is clear (Fig. 6d) that the hyper-Rec character of the double mutant is increased over that of the strains containing either single mutation alone. Again, similar results were obtained with the xseA8 mutation. We were able to obtain a quantitative measure of the hyper-Rec character of these strains by counting the papillae on a number of colonies. The results (see below) show that strain RS 5052 polAexl is approximately three times more hyper-Rec than its parent strain, KS439b, whereas strain KLC52 xseA7 is approximately two times more hyper-Rec than KS439b. The double mutant (KLC54 polAexl xseA7) is approximately two times more hyper-Rec than strain RS5052 polAexl and four times more hyper-Rec than strain KLC52 xseA 7. The specific

numbers of lac+ papillae estimated (± standard deviation) are as follows: KS439b polAex+ xse+, 6.6 ± 2.1; RS5052 polAexl xse+, 20.5 + 4.9; KLC52 polAex+ xseA7, 10.3 + 2.8; KLC54 polAexl xseA 7, 40.8 + 6.9. White (lac+) papillae on the surface of red (lacd) colonies were seen on lactose tetrazolium indicator plates. All strains were plated at the same time and incubated for 3 days at 300C. DISCUSSION In recent years several workers, including ourselves, have turned to mass screening procedures to isolate strains having reduced levels of nuclei acid enzymes in extracts where it has not been possible to predict a specific phenotype suitable for direct selection. This procedure has led to the isolation of mutants defective in DNA polymerase I (14), DNA polymerase II (8), exonuclease I (64), and exonuclease III (43). The successful isolation of mutants deficient in exonuclease VII reported in this paper provides one additional example of the feasibility of this approach. The enzymatic properties of exonuclease VII make it ideally suited for screening extracts of

944

CHASE AND RICHARDSON

large numbers of cells. The most important property in this respect is the full activity of the enzyme in the absence of added divalent cations (9), a property that distinguishes it from all other E. coli exonucleases. Using the procedures developed by Milcarek and Weiss (43), we have isolated -nine strains having reduced levels of exonuclease VII activity, five of which are of independent origin. Seven of the strains have little or no detectable exonuclease VII activity (2% of wild type or less), and one strain contains a temperature-sensitive activity. We have designated strains having reduced levels of exonuclease VII activity xse-. Mapping studies indicate that the defects in these strains occur at two or more loci, since xse- is 93 to 97% co-transducible with guaA in five of these strains but does not co-transduce with guaA at any detectable frequency in four of the strains. The high co-transduction frequencies of one group of strains with guaA make it reasonable to assure that the defects in these strains all occur within one structural gene for exonuclease VII, which we have designated xseA. One of the strains in this group (KLC3) appeared to contain a temperature-sensitive exonuclease VII activity in crude extracts, although the strain itself was not temperature sensitive for growth. The purification and study of the enzymatic properties of the exonuclease VII activity in this strain (KLC3) have confirmed that it is a thermolabile activity, and that xseA is a structural gene for exonuclease VII. It therefore is likely that all ofthe strains previously designated xseA - are defective in this gene, although this cannot be proven conclusively until genetic complementation analyses are performed. It seems most likely that the mutations that do not co-transduce with guaA occur at one or more other genes affecting the enzyme. Since exonuclease VII has not yet been purified to homogeneity, its subunit structure is unknown. It should also be pointed out that all of the xse- mutants we have isolated were obtained on the basis of having a reduced level of the 5' -* 3' hydrolytic activity of exonuclease VII, since the substrate used in the isolation was specific for this activity (see Materials and Methods and Results). The 3' -* 5' activity of the enzyme has not yet been measured in any of these mutants. The possibility exists, therefore, that some mutants may still contain the 3' 5' activity of exonuclease VII. During the course of our mapping studies we discovered that xseA - strains were more sensitive to the DNA synthesis inhibitor nalidixic acid (13, 21) than their parent strains. Al-

J. BACTERIOL.

though great effort has been expended in the study of nalidixic acid, the mechanism of its inhibition of DNA synthesis is unknown (5) at present. Two mutations in E. coli that confer resistance to various levels of nalidixic acid (23) have been identified and mapped. Mutations in the naiB gene result in resistance to low levels of the drug (4 Ag/ml), whereas mutations in the nalA gene result in resistance to high levels (80 ,ug/ml). Progress has recently been made by Sternglanz and his co-workers in the purification of the nalA gene product (R. Sternglanz, personal communication). It has also been shown by Hurwitz and his coworkers that nalidixic acid completely inhibits the replication of OX replicative form I DNA in an in vitro system (53). Mutants deficient in exonuclease VII may aid in deternining the mechanism of action of nalidixic acid, and this may in turn give insight into the role of exonuclease VII in DNA replication. We have observed no defect in the xseAmutants in their ability to grow phages T7, fd, and Ared. The ability of phage Ared mutants to grow on these strains is of interest, sincepolAex mutants do not plate Xr,d (27). The in vitro properties of exonuclease VII and the 5' -+ 3' activity of polymerase I suggest that these activities might have functions in vivo that overlap and therefore complement each other. However, it is clear that the 5' -- 3' activity of exonuclease VII does not serve the function of the 5' -* 3' activity of polymerase I in this case. Numerous models for recombination have been proposed requiring the specific removal of 3' or 5' single-stranded regions from intermediate recombinant molecules (11, 17, 50). These intermediates are, in effect, molecules with displaced single-stranded regions with either 3' or 5' termini. The in vitro properties of exonuclease VII suggested that it was ideally suited to perform this function, and therefore we proposed that it might play a role in recombination (10). Some of the studies undertaken in this preliminary characterization of the xseA - mutants have been specifically designed to test this possibility. It has been obvious from the beginning of our mapping studies that xseA mutants cannot be as recombination defective as many of the well-characterized rec mutants (12), since reasonable numbers of recombinants were obtained in the initial mating and transduction studies. To determine more carefully whether or not xseA - strains are recombination deficient, conjugational recombination proficiency was determined (Table 7). No evidence was found for any recombination defect in these mutants compared with their parent strain

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EXONUCLEASE VII MUTANTS OF E. COLI

945

when examined in this way. However, it is isolating and characterizing still other related clear that very small differences in recombina- activities and ultimately in defining specific tion frequencies (less than 5- to 10-fold) could pathways. The success of Clark and his coworkers in elucidating recombination pathnot be easily detected by this method. In a further effort to study the recombination ways in E. coli is extremely encouraging (12, properties of the xseA- strains, the hyper-Rec 26). Now that some basic insight into the funcphenotype ofthese mutants was examined. The tion(s) of exonuclease VII has been obtained, rationale for this approach was again that the future studies should define more clearly the 5' -- 3' hydrolytic activity of exonuclease VII role that this enzyme plays in DNA replication, might overlap and complement the 5' -. 3' recombination, and repair. activity of DNA polymerase I, and strains defective in the latter activity have been shown ACKNOWLEDGMENTS by Konrad and Lehman to be hyper-Rec (27). This investigation was supported by Public Health SerThe results of these studies (Fig. 6; see Results) grant AI-06045 from the National Institute of Allergy clearly show that the phenotype of strains defi- vice and Infectious Diseases, grant NP-1E from the American cient in exonuclease VII is similar to that of Cancer Society, Incorporated, and Public Health Service strains having reduced levels of the 5' -- 3' Research Career Development Award GM 13,634 (to C.C.R.) activity of DNA polymerase I. The exact inter- from the National Institute of General Medical Sciences. We are grateful to A. J. Clark and P. K. Storm for pretation of these results is, however, not yet allowing us to assay several of their recombination-deficertain. The level of increased recombination in cient strains for exonuclease VII. these strains is actually very slight compared to the wild-type strain (see Results) and may acLITERATURE CITED count for the fact that the recombination fre1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. quencies determined in Table 7 were not signifOptimal conditions for mutagenesis by N-methyl-N'icantly different from that determined from the nitro-N-nitrosoguanidine in Escherichia coli K12. wild-type strain. The molecular mechanism for Biochem. Biophys. Res. Commun. 18:788-795. the hyper-Rec effect is unknown, but possibly 2. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. any defect that prolongs the existence of single36:525-557. stranded regions in the DNA might result in a 3. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. hyper-Rec phenotype. On the molecular level, Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. the only defect that has been determined for S. D., H. Nagaishi, A. Templin, and A. J. hyper-Rec strains so far is in the joining of 4. Barbour, Clark. 1970. Biochemical and genetic studies of re"Okazaki fragments" and the accumulation of combination proficiency in Escherichia coli. HI. Rec+ very small DNA replicative intermediates (28). revertants caused by indirect suppression of Recmutations. Proc. Natl. Acad. Sci. U.S.A. 67:128-135. Whether xseA- strains share this defect with G. J., M. Levitt, and R. Sternglanz. other hyper-Rec strains is currently being in- 5. Bourguignon, 1973. Studies on the mechanism of action of nalidixic vestigated. acid. Antimicrob. Ag. Chemother. 4:479-486. Since we suggested that exonuclease VII 6. Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillamight function in recombination (10), several tion counter. Anal. Biochem. 1:279-285. new rec genes (recF, recG, recH, recJ, recK, 7. Campbell, J. L., H. Shizuya, and C. C. Richardson. recL) have been identified (26, 51). Two of these 1974. Mapping of a mutation, polBlOO, affecting degenes (recH and recJ) have been mapped in the oxyribonucleic acid polymerase II in Escherichia coli K-12. J. Bacteriol. 119:494-499. region of the chromosome near xseA, but at J. L., L. Soll, and C. C. Richardson. 1972. apparently different loci (26, 51). We have also 8. Campbell, Isolation and partial characterization of a mutant of shown that they all contain normal levels of Escherichia coli deficient in DNA polymerase II. exonuclease VII activity (Chase and RichardProc. Natl. Acad. Sci. U.S.A. 69:2090-2094. 9. Chase, J. W., and C. C. Richardson. 1974. Exonuclease son, unpublished data). There is, therefore, no VII ofEacherichia coli. Purification and properties. J. indication that any ofthese genes could be idenBiol. Chem. 249.-4545-4552. tical to any of the xse genes that we have identi- 10. Chase, J. W., and C. C. Richardson. 1974. Exonuclease fied so far. VII of Escherichia coli. Mechanism of action. J. Biol. Chem. 249:4553-4561. The complexity of defining the function(s) for A. J. 1971. Toward a metabolic interpretation of an activity for which no clear mutant pheno- 11. Clark, genetic recombination of E. coli and its phages. is the type exists apparent. However, imporAnnu. Rev. Microbiol. 25:437-464. tance of the problem is equally apparent. It is 12. Clark, A. J. 1973. Recombination deficient mutants of bacteria. Annu. Rev. Genet. 7:67-86. likely that activities of this type can only be 13. E. coliT. and other Cook, M., K. G. Brown, J. V. Boyle, and W. A. Goss. understood through the construction and study 1966. 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Escherichia coli mutants deficient in exonuclease VII.

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