JOUR1{NAI. OF BACTERIOLOGY, I)e. 1977, p. 8561-86(l Copyright (03 1977 American Society for Microbiology

Vol1. 1.322, No. 8 Pt rti tet l it7 l 'S. A.

Repair and Subsequent Fragmentation of Deoxyribonucleic Acid in Ultraviolet-Irradiated Bacillus subtilis recA C.

Uniuersity

1'. HADIDEN

Biomedical Sciences,

Ridge

Oak

and

Ridge National Laboratorv, Oak Ridge, Tennessee 37830

Biolog

Dici sion.

Received for publication :3l May 1977

Cells of Bacillus subtilis recAl are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system. Mutations resulting in recombination deficiency in bacteria often simultaneously affect the repair of damage to deoxyribonucleic acid (DNA). For example, recA, recB, and recC strains of Escherichia coli are sensitive to ultraviolet (UV) irradiation (25, 26) and X rays (15, 16). Of eight classes of recombination-deficient (Recd) mutants of Bacillus subtilis, coImprising mutations in at least 17 different genes, only two classes, with mutations in perhaps as few as two or three genes, show a normal resistance to irradiation or radiomirmetic agents (18). In general, it is not clear what steps in the repair/reconmbination processes are defective in the mutants. The mutation recA in B. subtilis 168 has an unusual pleiotropic effect on recombinational processes. Strains mutant in the recA gene are transformable at only 5 to 10'c of the normal level (9, 13, 14). W23 is a strain of B. subtilis related to strain 168. W2.3 strains are not transformable, but serve readily as donors in transformation of 168 strains (22). recA mutants of strain 168 are poorly transduced by phage PBS1 or SP10 lysates made in the related B. subtilis strain W23 (heterologous lysates), but they can be transduced at 50 to 60W of the normal level by PBS1 lysates made on derivatives of strain 168 (homologous lysates) (9, 14). They are also sensitive to UV irradiation (9) and mitomycin C (9, 14). This report presents evidence that DNA repair is not grossly defective immediately post-irradiation in this r ecA strain. However, sometime later, repaired sites are attacked by a nuclease or nucleases that cause breaks to reappear in the DNA at repaired sites without causing extensive degradation of DNA. Thus, radiation sensitivity and recombination deficiency in 856

a r ecA strain may both result from the defective nature of DNA repair. An accompanying paper ( 11) indicates that heterologous transforming DNA is incorporated but subsequently restricted in recA cells, implying that the defect in DNA repair may cause defective modification of newly synthesized DNA, so that the reappearance of breaks at repaired sites results from attack by restriction endonuclease.

MATERIALS AND METHODS Bacterial strains. Strains GSY 1025 (trpC2 nmetB4 recAl ), GSY1026 (trpC2 metB4), and GSY1027 (trpC2 metB4 uur-1) were obtained from J. A. Hoch (14). The prototrophic strain WB746 was obtained from E. W. Nester. Chemicals. [methyl- H Ithymicline ([VHld'T'hd, 44 Ci/mmll(ol) was obtained frormi Amnersham. [8- C ladenosine (58 nCi/ninoi) andl [ 'C luridine (204 m(i /mmol) were obtainetd from Schwarz/Mann., Orangeburg, N.Y. Hydroxylapatite chromatography was carried out onI Bio-Gel HTIP (Bio-Rad Laboratories, Richmond, Calif.), and 6-(p-hydroxyphenylazo)-uracil (Hl'UTra) was a generous gift from B. L. Langley, Imperial Chemical Co., Macclesfield, Enigland. Exonuclease III fromi F. (oli JG 112 (pol4) was purchased fromn Miles Lahoratories, Inc.. Elkhart, Ind., and was used without further purification. Growth of bacteria. UJnless otherwise noted, cells were grown at 37°C in mo(lified (3) Spizizen salts containing 0).5'r.c glucose, 0.01'i acid-hydrolyze(d casein (CAA, I)ifco, Detroit, Mich.), anti( 5)) pg each of niethioriniie and(l trvptophan per ml. P'reparation of tconpetenit cells and transformiationi were carried out as describedl p)reviously (12). For labeling of DNA with 'H, 2'-deoxyadenosine (30jitg/ml) and [HIdThd (20 pCi/ml) were added to the growth medium. Labeling with "C was accomplished by adding I pCi each of ['4Cluridline and ['4C]adenosine per ml. Colonv-forming units were enutimerate(d on I)ifco nutrient agar.

VOL. 132, 1977

857

BREAKAGE OF REPAIRED DNA IN recA B. SUBTILIS

Irradiation. Bacteria and transforming DNA were irradiated with UV as described previously (12). Xirradiation was done with a G. E. Maxitron 250 at a peak voltage of 250 kV and a current of 30 mA, with a 3-mm aluminum filter. The dose rate was 2,000 rads/min. UV-induced repair synthesis. Stationary-phase cells were irradiated and then diluted into growth medium containing (after dilution) 0.5% glucose, 0.01% CAA, tryptophan and methionine at 50 [Lg/ml each, 50 jig of 2'-deoxyadenosine per ml, 30 jCi of [3H]dThd per ml, and 300 jIM HPUra. These cultures were incubated at 37°C, and 50-tL samples were periodically removed. Precipitation and measurement of radioactivity in DNA were carried out as described by Billen and Hellermann (4). Cell titers were determined with a Petroff-Hausser counting chamber. Alkaline sucrose gradient centrifugation. Cells labeled with [3H]dThd were diluted after irradiation into fresh growth medium to a titer of about 5 x 107/ml. At appropriate intervals 200-lI samples were diluted with 200 L1I of ice-cold buffer containing 40 mM tris(hydroxymethyl)aminomethane (Tris) and 60 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.1. The cell suspensions were held in an ice bath until all samples had been taken. The cells were then pelleted by centrifugation, resuspended in 100 or 200 tL of a solution of 20 mM Tris and 30 mM EDTA (pH 8.1) plus 0.3 mg of lysozyme per ml, and incubated at 37°C for 3 to 4 min. This treatment left the cells essentially intact but fragile enough to be lysed by an alkaline solution. Samples of 50 jl (2.5 x 106 to 5 x 10"i cells) were layered on 5-ml linear density gradients (5 to 20%) of sucrose in 0.2 N NaOH, 0.5 M NaCl, and 1 mM EDTA (21), topped with a lysing layer of 100 jl of 0.2 N NaOH, 0.5 M NaCl, and 1 mM EDTA. The gradients were allowed to stand for an additional 20 min and then were centrifuged at 28,000 rpm for 90 min at 20°C with an SW50.1 rotor in a Beckman L5-50 preparative ultracentrifuge. Fractions were collected on Whatman no. 17 filter-paper strips, which were washed successively with 5% trichloroacetic acid and 95% ethanol before being counted by liquid scintillation spectrometry. Weight-average molecular weights (M,) were calculated by a computer program that was calibrated with several DNA species of known molecular weights. Induced breaks at time t were calculated as (2/M,), (2/Mw)n) uv; the number of noninduced breaks was about 10 breaks per 109 daltons of DNA. Exonuclease III assay of DNA ends. Cells were harvested by centrifugation, washed with sodium phosphate buffer (2 mM, pH 7.0), and resuspended in 2 ml of the same buffer plus 0.3 mg of egg white lysozyme per ml (Sigma Chemical Co., St. Louis, Mo.). After incubation at 37°C for 15 min, 6 ml of 8 M urea and 2 mM phosphate buffer was added. These lysates were bound to columns of hydroxylapatite (bed volume about 1.5 ml) equilibrated with 2 mM phosphate buffer and 6 M urea. The column was washed successively with 10 ml each of 2 mM and 0.1 M phosphate buffer. DNA was removed with 0.3 M phosphate buffer and collected in 1-ml fractions. The peak fractions, as indicated by radioactivity in the fractions, were com-

bined for the exonuclease III assay. The reaction mixtures contained 0.5 ml of DNA solution, 50 Id each of 150 mM MgCl2, 15 mM dithiothreitol, and water, and 100 pl of exonuclease III (32 U/ml in 0.05 M KCl, 0.1 mM EDTA, 1.0 mM dithiothreitol, 50% glycerol, and 0.01 M Tris [pH 7.4]). The mixtures were incubated at 37°C, and at 10-min intervals, 200-[.l samples were added to 50 [l of a solution of 5 mg of bovine plasma albumin per ml (Armour Pharmaceutical, Chicago, Ill.), followed by addition of 250 jl of 10% trichloroacetic acid. After 5 min of incubation in an ice bath, the tubes were centrifuged for 5 min at 5,;O00 rpm in the SS-34 rotor of a Sorvall RC2-B centrifuge. Duplicate 200-tl samples were mixed with 4.8 ml of a solution of 60 g of naphthalene, 8 g of Omnifluor (New England Nuclear, Boston, Mass.), 100 ml of methanol, and sufficient dioxane to bring the volume to 1 liter. Radioactivity was measured in an Intertechnique SL30 liquid scintillation spectrometer.

RESULTS Radiation sensitivity. The sensitivities to UV and X rays of strain GSY1025 (recAl) and its wild-type parent, GSY1026, are shown in Fig. 1. As had been shown previously (14), the recA mutation conferred marked sensitivity to UV. Strain GSY1025 was less UV sensitive than a Uvr- strain (GSY1027, Fig. 1A), unlike the recA mutants of E. coli (15, 16), which are clearly of a different nature than recA strains of B. subtilis. The recA strain has been shown to be sensitive to methyl methane sulfonate (13). It was also sensitive to X rays (Fig. 1B), as would be expected. Host cell reactivation of UV-irradiated trans1-

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UV FLUENCE (J/m ) X-RAY DOSE (krod) FIG. 1. Radiation sensitivity of Rec+ and recAl cells. Stationary-phase cells were irradiated as described in Materials and Methods. Surviving colonyforming units were enumerated on nutrient agar. (A) UV irradiated; (B) X-irradiated. Strains: (0)

GSY1026 (Rec+); (-) GSY1025 (recAl); (A) GSY1027 (uvr- 1).

8 58

HADDEN

J. BACTERIOL.

100forming DNA occurred in GSY1025. When the fluence responses of the inactivation of transforming DNA are compared, with strains 80GSY1025 and GSY1026 as recipients, there ap0 pears to be little difference in the rate of inactiO

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

JOUR1{NAI. OF BACTERIOLOGY, I)e. 1977, p. 8561-86(l Copyright (03 1977 American Society for Microbiology Vol1. 1.322, No. 8 Pt rti tet l it7 l 'S. A...
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