MGG

Molec. gen. Genet. 168, 37-47 (1979)

© by Springer-Verlag 1979

Introduction of an Active Enzyme into Permeable Cells of Escherichia coli Acquisition of Ultraviolet Light Resistance by

uvr

Mutants on Introduction of T4 Endonuclease V

Kenji Shimizu and Mutsuo Sekiguchi Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan

Summary. Plasmolysed cells of Escherichia coli N212 (uvrA recA) acquired ultraviolet resistance when the cells were exposed to high concentrations of T4 endonuclease V. With increasing concentrations of T4 enzyme, survivals of plasmolysed cells after ultraviolet irradiation increased while colony-forming ability of unirradiated plasmolysed cells was not significantly affected by the enzyme treatment. Under appropriate conditions more than 200 fold increase in survivals was observed. When plasmolysed cells were treated with a pre-heated enzyme preparation or enzyme fractions derived from T4v 1 (endonuclease V-deficient mutant)-infected cells, only little or no reactivation took place. Permeabilization of cells prior to the enzyme treatment was essential for the effective reactivation. Treatment of intact cells with the T4 enzyme did not cause any reactivation. Cells treated with 20 m M E G T A or 50 m M CaCI 2 in cold were reactivated to certain extents by the enzyme, but the extents of the reactivation were far less compared to those of plasmolysed cells. Plasmolysed cells of strains carrying a mutation in one of uvrA, uvrB and uvrC genes were reactivated by introduction of T4 endonuclease V, as was the uvrA recA double mutant. UvrD mutants were also reactivated, but rather slightly. However, wild type strain as well as strains having a mutation in recA or polA gene were not reactivated. F r o m these results it was suggested that T4 endonuclease V, taken up into permeable cells, can function in vivo to replace defective functions, which are controlled by the uvr genes. The conditions established in the present For offprints contact: Dr. K. Shimizu

study may be used for introduction of other proteins into viable bacterial cells.

Introduction Artificial introduction of biologically active protein molecules into a living cell system should provide useful means for analysis of cellular functions and also for assay of certain proteins whose activity cannot be measured by ordinary biochemical methods, A major obstacle in this approach is impermeability of the cell membrane to large molecules. Several attempts have been made to introduce macromolecules into animal cells (Papahadjopoulos et al., 1974; Furusawa et al., 1974; Loyter et al., 1975). These depended on the interaction of the cell membrane with liposomes or with HVJ (Sendai virus). Evidence was recently presented that an active enzyme inserted into human cultured cells by the action of HVJ can function in situ (Tanaka et al., 1975, 1977). Although many methods to permeabilize bacterial cells have been presented (Leive, 1965; Buttin and Kornberg, 1966; Moses and Richardson, 1970; Vosberg and Hoffmann-Berling, 1971 ; Wickner and Hurwitz, 1972; Moses, 1972), most of these treatments are too drastic to preserve colony-forming ability of the cells. No evidence has been presented to show that a functional enzyme is artificially inserted into viable bacterial cells. We were thus led to explore the possibility of making permeable bacterial cells which still retain cell viability and introducing a biologically active protein into such cells.

0026-8925/79/0168/0037/$02.20

38

K. Shimizu and M. Sekiguchi : Introduction of Enzyme into Permeable Cell

As a protein to be tested for insertion, we have chosen T4 endonuclease V, a bacteriophage T4-coded repair enzyme (Yasuda and Sekiguchi, 1970b). The enzyme is small (molecular weight, 18,000) and has a high specificity for ultraviolet light (u. v.) 1_damaged DNA. It has been shown that the enzyme introduces a single-stranded break at the Y-side of a pyrimidine dimer and is responsible for the first step of excision repair in T4-infected cells (Sekiguchi et al., 1975). It was assumed that effective introduction of the enzyme into cells of Escherichia coli mutants which are defective in a similar enzyme activity should increase survivals of the cells after u. v. irradiation. This should be a sensitive measurement for insertion of a functional enzyme into viable cells. To establish conditions for introduction of an enzyme into viable cells, we have prepared various permeable cell preparations and have examined their response to T4 endonuclease V treatment. It was found that plasmolysed cells, prepared with the use of an appropriate sucrose concentration, of certain u. v.sensitive mutants acquire u. v. resistance on exposure to the T4 enzyme. We have also explored the usefulness of this system for identifying functions of certain E. coli gene products in D N A repair. A preliminary account of parts of these experiments has been reported (Shimizu and Sekiguchi, 1977).

Table 1. E. coli strains used

Strain

Relevant genotype

Source and reference a

W3623

trp, gal, strA

K. Shimada (1)

N17-9

as W3623 but uvrA54

K. Shimada (1)

N3-1

as W3623 but uvrB5l

K. Shimada (1)

N12-2

as W3623 but uvrC53

K. Shimada (1)

N17-2

as W3623 but uvrC55

K. Shitnada (1)

N14-4

as W3623 but uvrD3

K. Shimada (1)

N13-I

as W3623 but uvrD2

K. Shimada (1)

N611

as W3623 but polA

H. Ogawa (2)

N212

uvrA54, recA1

A. Miura (3)

KL16-99

recA1 (thi-1, reLl-drm-3)?

N. Otsuji (4)

AB 1 157

thr-1, leu-6, proA2, his-4, argE3, thi-1, lacY1, galK2, ara-14, xyl-5, mtl-1, tsx-33 strA31, supE44

N. Otsuji (5)

AB1886

as ABl157 but uvrA6

N. Otsuji (5)

AB1885

as ABl157 but uvrB5

N. Otsuji (5)

AB1884

as ABl157 but uvrC34

N. Otsuji (5)

11O0

endA ( thi-1, sup-44) ?

H. HoffmannBerling (6)

a Numbers in parenthesis denote following references: (1) Ogawa et al. (1968); (2) Ogawa (1970); (3) Miura and Tomizawa (1968); (4) Low (1968); (5) Howard-Flanders et al. (1966); (6) Diirward and Hoffmann-Berling (1968).

Materials and Methods c) Enzymes a) Bacteria and Phage Strains The strains of bacteria used in these experiments are listed in Table 1. Bacteriophage T4D and T4vl, a mutant defective in T4 endonuclease V activity (Harm, 1963; Yasuda and Sekiguchi, 1970a), were originally obtained from W. Harm.

b) Media L-broth contains, per liter of distilled water, 10 g polypeptone, 5 g yeast extract, 5 g NaC1 and 1 g glucose. The composition of TG medium was as described (Wickner and Hurwitz, 1972). M9S contains, per liter of distilled water, 5.8 g NazHPO4, 3 g KH2PO4, 5 g NaC1 and 1 g NH4C1. BSSE (balanced salt solution supplemented with EGTA) contains 0.14M NaC1, 5.4raM KC1, 0.34mM Na2HPO4, 0.44ram KH2PO~, 10mM Tris . HC1 (pH 8.0) and 2 m M ethylene glycol-bis-(fl-aminoethyl ether) N, N'-tetraacetic acid (EGTA).

Abbreviations used." BSSE, balanced saIt solution containing EGTA; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis-(fl-aminoethyl ether) N, N'-tetraacetic acid; PEME, 10 m M potassium phosphate (pH 6.5)-10 m M 2-mercaptoethanol10% ethylene glycol-2 m M EDTA; SDS, sodium dodecylsulfate; u. v., ultraviolet light; vR, reactivation mediated by T4 endonuclease V (the v gene product)

T4 endonucIease V was prepared from T4D-infected E. coli 1100 as described (Yasuda and Sekiguchi, 1976). The purification procedure included phase partition in dextran-500/polyethylene glycol6000 (Fraction II), column chromatography on CM-Sephadex C-25 and twice repeated chromatography on hydroxylapatite. The second hydroxylapatite fraction (Fraction V, 38 units/74 pg protein/ ml), purified about 700 fold over the extract, was used in most of these experiments. A SDS-polyacrylamide disc gel electrophoresis of Fraction V revealed that the purity of T4 endonuclease V was approximately 40%. The enzyme was used after dialysis at 0 ° C for 6 h against 1,000 volumes of BSSE in order to remove EDTA which inhibits growth of E. coli cells. For the experiments described in the third part of Results, enzyme fractions were prepared from T4D-infected and T4v 1infected E. coli 1100 by a slight modification of the original procedure. After loading of Fraction II onto a CM-Sephadex C-25 column (1 x 15 cm), the column was washed with 60 ml PEME buffer (10 m M potassium phosphate (pH 6.5)-10 m M 2-mercaptoethanol-10% ethylene glycol-2 m M EDTA) containing 0.15 M KC1 and then eluted with the same buffer containing 0.35 M KCI. Five ml fractions were collected. The enzyme activity was determined by measuring degradation of 32p-labeled, u. v.-irradiated T4 D N A in the presence of T4vlinfected cell extract. One unit of endonuclease V is defined as the activity that releases 24 nmol of nucleotides as acid-soluble material in 20 min at 37 ° C (Yasuda and Sekiguchi, 1970b; 1976). Protein was determined by the method of Lowry et al. (1951).

K. Shimizu and M. Sekiguchi: Introduction of Enzyme into Permeable Cell

d) Chemicals Sucrose and SDS were of specially prepared reagent grade for ultracentrifugation and for electrophoresis, respectively, and were purchased from Nakarai Chemical Co. Ltd. CM-Sephadex C-25 was purchased from Pharmacia Fine Chemicals, and hydroxylaparite, Hypatite C, from Clarkson Chemical Co. Inc. A synthetic penicillin was obtained from Takeda Chemical Industries.

e) Ultraviolet Light Irradiation In most experiments, cells were diluted to 4 x 107 cells/ml in M9S and irradiated with a 15 W Toshiba germicidal lamp at room temperature. Irradiation was done at a distance of 94 cm from the lamp (dose rate: 0.5 j/m2/s). The dose rate was measured by a Blak-ray J-255 shortwave u. v. intensity meter. In the case of E. coil N212 (uvrA recA), the distance was 214cm (dose rate: 0.06 j/m2/s) and the dose rate was calibrated by measuring survivals of E. coli N212 cells under the two conditions. To avoid photoreactivation, all operations were carried out in the dark or under a yellow dim light.

f) Preparation of Plasmolysed Cells Unless otherwise noted, cells were grown in L-broth at 37° C and harvested during the late logarithmic phase of growth. Cells were collected in a refrigerated centrifuge and washed once with cold TG medium. The cells were finally suspended in ice-cold plasmolysing buffer at a cell density of 5x I09/ml and kept at 0°C for 30 min. The plasmolysing buffer (Wickner and Hurwitz, 1972), which contains 1.98 M sucrose-10 mM EGTA-40 mM Tris.HC1 (pH 8.0), was prepared immediately before use by mixing 1 part of 100mM EGTA, 1 part of 400raM Tris-HCl (pH 8.0) and 8 parts of saturated sucrose solution (at 0° C).

g) Reactivation A portion (5 gl) of a suspension of permeabilized cells was added to 50 gl of BSSE containing T4 endonuclease V, After standing at 0°C for 30 min, the cell suspension was diluted 10 fold with M9S (at room temperature). Portions (0.2 ml each) of the diluted cell suspension were transferred to holes (7 mm in diameter) of a microtitre plate (Cooke Engineering Co.) and irradiated with u. v. In some experiments, cells were preirradiated, permeabilized and then treated with T4 endonuclease V. Unirradiated or irradiated samples were diluted with M9S and plated on L-broth agar plates. Usually three determinations were carried out for each sample. After incubation at 37° C for 30 h in the dark, number of colonies formed was counted. Reactivation index (fold) was expressed as a ratio of surviving fraction of enzyme-treated cells to that of untreated cells. Since values of the index varied according to u.v. doses, the fraction of annulment of u.v. doses by the enzyme treatment was employed to evaluate the efficiency of reactivation.

39

carrying m u t a t i o n s in b o t h uvrA a n d recA genes were used as a test strain. The d o u b l e m u t a n t is extremely sensitive to u.v., a n d it has been s h o w n that such a m u t a n t is killed by f o r m a t i o n of a single p y r i m i d i n e dimer in the g e n o m e whereas a b o u t 3200 dimers are needed to p r o d u c e one lethal hit in wild type strain ( H o w a r d - F l a n d e r s et al., 1969). Thus, repair of a few dimers m a y be sufficient to recover the m u t a n t cell f r o m u . v . - i n d u c e d cell killing, a n d this s h o u l d provide a sensitive assay system for i n t r o d u c t i o n of a functional repair enzyme into cells. E. coli N212 (uvrA54 recA1) was g r o w n in L - b r o t h a n d harvested d u r i n g the late l o g a r i t h m i c phase of growth. Cells were subjected to various t r e a t m e n t s which are k n o w n to increase p e r m e a b i l i t y to large or charged molecules. These t r e a t m e n t s i n c l u d e d plasmolysis of cells with a high c o n c e n t r a t i o n of sucrose ( W i c k n e r a n d H u r w i t z , 1972), t r e a t m e n t with E D T A or E G T A (Leive, 1965), t r e a t m e n t with 0.05 M CaC12 ( M a n d e l a n d Higa, 1970) a n d exposure to a low c o n c e n t r a t i o n of SDS (Otsuji et al., 1972). The treated cells retained most of their ability to p r o d u c e colonies on n u t r i e n t agar, a n d even after storage at 0 ° C for 18 hr the treated cell p r e p a r a t i o n s exhibited m o r e t h a n 10% of the original viability. O n the other h a n d , spheroplasts, p r o d u c e d b y lysozyme ( Z i n d e r and A r n d t , 1956) or penicillin t r e a t m e n t (Lederberg a n d Clair, 1958), were practically n o n - v i a b l e (less t h a n 0.1% of the original viability), as expected from their lack of cell wall structure. To c o m p a r e abilities of p e r m e a b l e cells to take u p an exogenous enzyme, each cell p r e p a r a t i o n was exposed to T4 e n d o n u c l e a s e V for 30 m i n at 0 ° C and then the u. v. sensitivity was determined. As a c o n t r o l each p r e p a r a t i o n was treated in a similar m a n ner except the omission of the enzyme. W e calculated the r e a c t i v a t i o n i n d e x b y dividing the surviving fraction (ratio of the n u m b e r of colonies of u. v.-irradiated sample to that of n o n - i r r a d i a t e d sample) of enzymetreated cells by the suviving fraction of n o n - t r e a t e d cells. As can be seen f r o m T a b l e 2, the highest reactivat i o n index was o b t a i n e d with plasmolysed cells. Cells treated with E G T A or CaC12 were reactivated to certain extents, b u t their levels were low c o m p a r e d to that of plasmolysed cells. Other p r e p a r a t i o n s , as well as intact bacteria, were n o t reactivated by the enzyme.

Results

b) Conditions for Reactivation a) Response of Permeable Cells to T4 Endonuclease V I n initial experiments to explore c o n d i t i o n s for introd u c i n g a n e n z y m e into viable cells, an E. coli strain

Of the p e r m e a b l e cell p r e p a r a t i o n s tested, plasm o l y s e d cells appear to be m o s t suitable for the present purpose. Thus, o p t i m a l c o n d i t i o n s for the reactiv a t i o n were studied with this system.

40

K. Shimizu and M. Sekiguchi : Introduction of Enzyme into Permeable CelI

Table 2. Effect of various treatments on the reactivation of u. v.-irradiated cells by T4 endonuclease V Treatment

Viability (%) 0h

None" Plasmolysisb

100 35.9

18 h

T4 u.v. endo- surving nucle- fraction ase V

Reactivation index (fold)

96.6

+

9.07 x 10 3 9.32x10 3

1.0

22.0

+

3.95 x 10- 3 3.12x 10 -z

7.9

20 m M E G T A c

94.4

69.1

+

2.40 x 10 3 9.80x 10 -3

4.1

10 mM EDTA a

72.6

3.7

+

9.82 x 10 3 9.16x 10 -3

0.9

50 mM CaC12 e

83.6

12.8

+

1.52 x 10-2 3.39 x 10 -2

2.2

+

1.95 x 10 a 3.19x10 a

1.6

+

2.08 x 10- 1 2.50 x 10-1

1.2

+

1.26 x 10 -1 2.02 x 10-1

1.6

0.1% SDS J"

52.0

Penicillinspheroplastsg

0. I

Lysozymespheroplastsh

0.17

13.8

E. coli N212 (recA1 uvr A54) cells, grown in L broth at 37°C, were harvested during the late logarithmic phase of growth, washed once, and suspended in cold TG medium at a cell density of 4.5 x 109/ml. Portions of cells were spun down and treated as follows; (a) resuspended in TG medium; (b) plasmolysed as described in Materials and Methods; (c) treated with 20 mM EGTA in 40 mM Tris-HC1 (pH 8.0) at 0 ° C for 30 rain; (d) treated with 10 m M EDTA in 40 mM Tris. HC1 (pH 8.0) at 0 ° C for 30 rain; (e) treated with 5 0 m M CaCI2 at 0°C for 30min; (J) treated with 1 mg/ml of SDS at 0 ° C for 30 rain, washed, and resuspended in cold TG medium; (g) aerated overnight at 37 ° C in sucrosebroth containing 125 gg/ml of penicillin and resuspended in TGmedium containing 10% sucrose; (h) incubated at 30 ° C for 15 rain in 1.5 M sucrose-10 m M EGTA-10 mM Tris.HC1 (pH 8.0)-100 gg/ ml of lysozyme (after chilling MgSO4.7Hz0 was added to give a final concentration of 4%). Volumes of all preparations were adjusted to those of the initial cell suspension. Numbers of viable cells were determined immediately after each treatment or after storage at 0 ° C for 18 h. To 5 ~tl of each preparation 50gl of BSSE with or without T4 endonuclease V (3.8 units/ml) were added. The mixture was stood in ice for 30 min, diluted, and the u. v. sensitivity was measured as described in Materials and Methods. U. v. fluences were 0.4 J/m z for samples (a) to (f) and 0.2 J/m 2 for (g) and (h)

It was found that the growth conditions of bacteria are important for obtaining competent plasmolysed cells. As shown in Table 3, bacteria grown in L-broth and harvested during the late logarithmic phase or stationary phase yielded more competent cells compared to bacteria grown in the same medium and harvested during the mid-logarithmic phase. Cells grown in DC medium (Wickner and Hurwitz, 1972)

Table 3. Effect of growth phase of celis on efficiency of reactivation Expt. No.

Growth phase of cells

Turb- Surviving fraction idity - Enzyme + Enzyme

Reactiration index

(fold) I. 2. 3.

Logarithmic 0.50 Late-logarithmic 0.74 Stationary 0.95

1.3x10 -4 2.0 x 10-4 1.9x 10-5

6.1x10 4 4.4x 10 3 3.3× 10 4

4.7 22.0 17.4

Plasmolysed cells were prepared from cultures of E. coli N212 harvested at different phases of growth. The reactivation experiments were performed using 25 units/ml of T4 endonuclease V (see Materials and Methods). U. v. fluence was 0.5 J/m z for Experiments 1 and 2, and 0.6 J/m 2 for Experiment 3

Table 4. Effect of temperature on the reactivation of cells by T4 endonuclease V Temperature

0° C 30 o C

Surviving fraction - Enzyme

+ Enzyme

Reactivation index (fold)

2.3x10 4 2.3 × 10 -4

6.8x10 3 1.3 x 10 -3

29.6 5.7

A suspension of playsmolysed cells of E. coli N212 was prepared as described in the legend to Fig. 1. To 5 gl of the cell suspension, 50 gl of BSSE with or without T4 endonuclease V (25 units/ml) were added at 0° C. The mixture was incubated for 30 rain at the indicated temperatures, diluted and the sensitivity to u. v. was measured. U. v. fluence used was 0.5 J/m 2

were as competent as those grown in L-broth provided that the cells were harvested during the late logarithmic phase and plasmolysed under the standard condition. When bacteria were grown in M9 medium enriched with 1% polypeptone, less competent plasmolysed cells were obtained. A buffer containing 1.98 M sucrose was found to be most suitable for preparing competent plasmolysed cells. With the use of this buffer plasmolysed cells which retained 5 to 45% of original viability were usually obtained. At a higher sucrose concentration (2.2 M), less than 1% of plasmolysed cells were viable. When lower sucrose solution (e.g. 1.8 M) was used, less competent cells were obtained. We then examined conditions for treatment of plasmolysed cells with T4 endonuclease V. As shown in Table 4, a higher level of reactivation took place when plasmolysed cells were exposed to the enzyme at 0°C rather than at 30 ° C. Similar effects of low temperature have been observed with CaC12- and EDTA-treated cells, in which efficient penetration of D N A or nucleotides occurred at low temperature (Leive, 1965; Mandel and Higa, 1970). Finally the time of treatment of plasmolysed cells with the en-

K. Shimizu and M. Sekiguchi: Introduction of Enzyme into Permeable Cell

41 I

~J -'~

1oo v ~11)

~" " . .... . ....................................................................

u v

30

50

u

5

I

I

~ -.o.....o.......

[n

150

9

100

'13

-iJ

30 u rd L_

50

4

L 30

1o E

"T C O c

L-

20

i0 7J

3

.,-

_a r~ r0

2

/3 r~

1

:2,

".C

.-

>

rd

Z ,g .>

1

ID 1>

u nn--

0

5

10

20

T4 E n d o n u c [ e a s e

40

n,"

V (units/m[)

Fig. l. Effect of T4 endonuclease V on u. v. resistance of plasmolysed or EGTA-treated cells. E. coli N212 cells were grown in L-broth to an optical density (660 rim) of 0.9 and plasmolysed by suspending in 1.98 M sucrose-10 mM EGTA-40 mM Tris. HC1 (pH 8.0) and keeping at 0° C for 30 min. EGTA-treated cells were prepared by suspending in 10 mM EGTA-40 mM Tris.HC1 (pH 8.0) at 0° C for 30 min. To 5 lal of the permeable cell suspension were added 50 lal of BSSE containing various concentrations of T4 endonuclease V, and the mixture was stood in an ice-bath for 30 min. After 10 fold dilution with M9S, u. v. sensitivity of the cells was measured as described in Materials and Methods. (o) Plasmolysed cells, treated with enzyme; (o) EGTA-treated cells, treated with enzyme; (x) plasmolysed cells, treated with heated enzyme (45° C for 20 min). Although the heated enzyme possessed no endonuclease activity, the data were plotted against the initial activity before heating. (zx) Relative viability of unirradiated, plasmolysed cells after incubation with T4 endonuclease V

zyme was optimized to 30 m i n at 0 ° C ; t r e a t m e n t for times shorter t h a n 15 m i n or longer t h a n 45 rain caused lower levels of r e a c t i v a t i o n (data n o t shown).

c) Dose-Response Relationship By using the c o n d i t i o n s established in the preceding section, correlation b e t w e e n the a m o u n t s of T4 endonuclease V used a n d the levels of r e a c t i v a t i o n was investigated. W h e n plasmolysed cells of E. coli N212 were treated with various a m o u n t s of T4 enzyme a n d then irradiated with u, v., the result s h o w n in Fig. 1 was obtained. W i t h increasing c o n c e n t r a t i o n s of the enzyme, survivals of u. v.-irradiated cells increased while n u m b e r s of u n i r r a d i a t e d cells decreased slightly. Levels of the r e a c t i v a t i o n were almost p r o p o r t i o n a l to the a m o u n t s of the enzyme applied, a n d m o r e t h a n 30 of r e a c t i v a t i o n index was o b t a i n e d at 38 units/ ml of the enzyme. Significant b u t low levels of reactiv a t i o n t o o k place w h e n E G T A - t r e a t e d cells were ex-

O

, 5

I 10

I 20

I ] 40

[K

T4 E n d o n u c L e a s e V ( u n i t s / m [ )

Fig. 2. Reactivation of u. v.-irradiated, plasmolysed cells by T4 endonuclease V. Cells were suspended in cold TG medium at 2.5 x l0 s cells/ml and irradiated with about 0.5 J/m z of u. v. The bacteria were spun down, resuspended in one tenth volume of the plasmolysis solution and kept in ice for 1 hr. Non-irradiated cells were plasmolysed in a similar manner. To 5 gl of irradiated or non-irradiated samples were added 50 gl of BSSE containing various amounts of T4 endonuclease V. After standing in ice for 30 min, the suspension was diluted and plated. (e) Relative viability of irradiated cells; (©) relative viability of nonirradiated cells

posed to the T4 e n z y m e ; the reactivation index at the same high level of the enzyme was only 3. N o reactivation was i n d u c e d when the plasm o l y s e d cells were treated with an enzyme preparation which h a d been i n c u b a t e d at 4 5 ° C for 20 m i n (Fig. 1). Since T4 e n d o n u c l e a s e V is a t h e r m o l a b i l e p r o t e i n a n d the activity is almost completely inactivated by such t r e a t m e n t (Sato a n d Sekiguchi, 1976), it is likely that the reactivation is caused b y T4 endonuclease V per se, b u t n o t other factor(s) which m i g h t be present in the enzyme p r e p a r a t i o n . T o exclude a possibility that an a p p a r e n t increase in the u. v. resistance of the enzyme-treated, plasmolysed cells is due to shielding effect of protein, a following experiment was performed. Bacteria were first irradiated with u. v. a n d then plasmolysed a n d treated with the T4 enzyme. I n this case, too, there was a net increase in survivals of irradiated cells (Fig. 2). It is evident, therefore, that u. v . - d a m a g e d cells are actually reactivated by the enzyme t r e a t m e n t . The lower degree of r e a c t i v a t i o n observed in this experiment as c o m p a r e d to that of the previous one m a y be due to d e g r a d a t i o n of D N A in irradiated cells before the enzyme action. It has been shown that extensive d e g r a d a t i o n of D N A takes place in recA uvrA cells after u. v . - i r r a d i a t i o n ( H o w a r d - F l a n ders et al., 1969).

42

K. Shimizu and M. Sekiguchi : Introduction of Enzyme into Permeable Cell i

r

J

I

r

i

I

,

U.V.

I

(b)

(o)

0 1 IL

30

0.2 I

dose 0.4 I

(J/m 0.6 E

2) 1.0

0.8 I

E 3 "IJ

/\

20

10-I i

5O

o n~

40

o

t/I

10 -2

vR

30 0.5

l

o10 n~

o

20

n-

;

,~,~________,~,, /

i

I

,

~

5

,

,

,

i

l

I0 Fraction

"~

1 5 number

"~..A

10

o -o c bJ

10

Fig. 3a and bo Effect of enzyme fractions from T4D-infected and T4vl-infected E. coli on the reactivation. F r o m 24 g each of T4Dinfected and T4vl-infected E. coli 1100 cells, extracts were prepared and processed as described in Materials and Methods. Fractions eluted from CM-Sephadex C25 columns were dialysed against BSSE at 0 ° C for 6 h, diluted 10 fold with cold BSSE and then applied to plasmolysed cells of E. coli N212. Treated cells were irradiated with u. v. (0.5 J / m 2) and the reactivation index was determined, a Fractions from T4D-infected cells, b Fractions from T4vl-infected cells. (o) Reactivation (fold); ( e ) T4 endonuclease V activity; (zx) protein concentration

d) Specific Action of T4 Endonuclease V It has been shown that synthesis of T4 endonuclease V is controlled by the v (den V) gene of T4 and that T4vl m u t a n t is unable to induce the enzyme synthesis (Harm, 1963; Y a s u d a and Sekiguchi, 1970a). To show u n a m b i g u o u s l y the involvement of the T4 enzyme in the reactivation process, we have c o m p a r e d effects of enzyme fractions f r o m T 4 D (wild type)infected and T4vt-infected cells. A n extract of T4vlinfected cells contained other T4-induced enzyme activities, such as exonuclease B and C (Shimizu and Sekiguchi, 1976), but not T4 endonuclease V. Extracts of the two types of infected cells were processed in similar m a n n e r s to the step of CM-Sephadex c h r o m a t o g r a p h y . As shown in Fig. 3, elution profiles of protein were essentially the same for the two samples; however, only little or no T4 e n d o n u clease V activity was f o u n d in the fractions of T4v 1infected cells. W h e n the fractions derived f r o m T4D-infected cells were applied to plasmolysed cells, significant levels o f activity that recovers the cells f r o m u. v.induced damages were detected. The levels of the in vivo reactivation activity coincided well with the levels of the endonuclease activity determined in vitro. On the other hand, only little reactivation was induced by the fractions derived f r o m T4vl-infected cells. Thus, it is evident that T4 endonuclease V, the v gene product, is indeed responsible for the reactivation.

(0.54)

io-3 (o.34) ¢.,

L u3

I

I

1

1

Fig. 4. Acquisition ofu. v. resistance by E. colicells on introduction of T4 endonuclease V. Plasmolysed cells of E. coli N212 were swollen in BSSE with or without T4 endonuclease V at 0° C for 30 rain. Cells were diluted 10 fold with M9S and then irradiated with u. v. at various doses. Results of several independent experiments are illustrated. (e) Cells treated with BSSE without enzyme; (~) cells treated with 38 units/ml of enzyme; (o) cells treated with 25 units/ml of enzyme. Horizontal lines and vR are explained in the text

e) Acquisition of U. V. Resistance Figure 4 shows u. v. survival curves of plasmolysed cells o f E. coli N212 (uvrA recA) which had been treated with two different levels of T4 endonuclease V. The result indicates clearly that plasmolysed cells acquire u. v. resistance on introduction of T4 enzyme. U. v. resistance levels of the enzyme-treated cells were higher than those of untreated cells over the wide ranges of u. v. doses examined. As high as 200 fold reactivation for irradiation at 1 J/m 2 was observed with plasmolysed cells treated with 25 units/ml of the enzyme. A n o t h e r notable feature is the observation that the survival curves for N212 cells treated with the enzyme are u p w a r d l y concave, indicating that m o r e efficient reactivation takes place when the cells have been irradiated with higher doses of u. v. Since a similar concave curve has been obtained with recA m u t a n t while the u. v. sensitivity curve of uvrA m u t a n t is rather straight (Clark and Margulies, 1965; H o w ard-Flanders et al., 1966), this result is consistent with the idea that the uvrA function but not the recA function is replaced by the T4 enzyme, as will be shown below.

K. Shimizn and M. Sekiguchi : Introduction of Enzyme into Permeable Cell U.V. 0

50

100

J/m 2 )

dose m

10

20

1

1

),,

1G1

43

10

10

0

20

Cb)

xK

o~

.; io-3 "g

60

'

' (bl

I

I

vR (0.39)

10-2 O

( J / m 2) 20 40

_•,,,,

10-~

',,q

102

U.V. dose 20 0 I (a)

vR 10-3

2 \

t/3

10-4 I

E

164

Fig. 5a-c. Specific reactivation of uvrA mutant by treatment with T4 endonuclease V. Experiments were carried out as described in the legend to Fig. 4, using plasmolysed cells of several E. coli strains, a W3623 (uvr+, rec+), b N17-9 (uvrA54); c KL16-99 (recA1). (o) Treated with T4 endonuclease V (25 units/ml); (o) not treated with the enzyme

f ) Complementation of uvr Functions with T4 Enzyme A p p l i c a t i o n of T4 e n d o n u c l e a s e V to p l a s m o l y s e d cells o f E. coli W3623 (uvr + rec +) d i d n o t cause a n y increase in the u. v. resistance (Fig. 5a), which is consistent with the n o t i o n t h a t the T4 e n z y m e substitutes for a function(s) t h a t is missing in N212 cells. T o see which of the functions, c o n t r o l l e d b y the gene uvrA or recA, is r e p l a c e d b y the enzyme, e x p e r i m e n t s were carried out with strains c a r r y i n g either one of the m u t a t i o n s . As s h o w n in Fig. 5 b a n d c, the u. v. resistance level o f N17-9 (uvrA54) increased c o n s i d e r a b l y after t r e a t m e n t with the e n z y m e whereas the s a m e t r e a t m e n t d i d n o t cause a n y increase for K L 1 6 - 9 9 (recA1) cells. F r o m the curves o f Fig. 5 b it was e s t i m a t e d t h a t a p p r o x i m a t e l y 50% o f the lethal lesions in irrad i a t e d uvrA cells were r e p a i r e d b y the T4 e n z y m e

I

(c) L-

-1

10 -

*",

iD

•

10-2 ~

vR

" ~ o 1

10-3 10-L'

Since the r e a c t i v a t i o n index changes with the u. v. doses used, the efficiency of r e a c t i v a t i o n was estim a t e d as follows. The h o r i z o n t a l lines d i r e c t e d f r o m the p o i n t s on the survival curves of e n z y m e - t r e a t e d cells at 0.7 J / m 2 t o w a r d the o r d i n a t e axis p e r m i t us to calculate the efficiency o f r e a c t i v a t i o n (vR sector). T h e sections b e t w e e n the p o i n t s and the curve o f n o n - t r e a t e d cells r e p r e s e n t the fractions of the u. v.i n d u c e d lethal d a m a g e s t h a t are in effect a n n u l l e d by vR, whereas the t o t a l lengths o f the h o r i z o n t a l lines r e p r e s e n t the t o t a l lethal d a m a g e s in each cell. T h e ratios i n d i c a t e t h a t at least 34% a n d 54% of the lethal lesions in E. coli N212 cells are r e p a i r e d b y t r e a t m e n t with 25 u n i t s / m l a n d 38 u n i t s / m l of T4 e n d o n u c l e a s e V, respectively.

I

0

I 20

1 40 0

I

20

4;

Fig 6a-d. Effect of T4 endonuclease V on the u. v. sensitivity of several repair-defective mutants of E. coli. Experiments were done as described in Fig. 4. a uvrB mutant; N3-1 (uvrB51). b uvrC mutants; N12-2 (uvrC53) for circles, N17-2 (uvrC55) for triangles, c uvrD mutant; N14-4 (uvrD3). d polA mutant; N611 (polA). (o, ~) Treated with T4 endonuclease V (25 units/ml); (o, A) not treated with enzyme

i n t r o d u c e d (25 units/ml). A n o t h e r n o t a b l e f e a t u r e of this e x p e r i m e n t is t h a t the efficiency o f r e a c t i v a t i o n of uvrA cells is c o n s t a n t over different u. v. doses, u n l i k e the r e a c t i v a t i o n o f uvrA recA cells. Effectiveness of the e n z y m e t r e a t m e n t was tested for o t h e r E. coli m u t a n t s which are k n o w n to be defective in the process of excision r e p a i r ; n a m e l y , uvrB, uvrC, uvrD a n d polA m u t a n t s . As s h o w n in Fig. 6, an efficient r e a c t i v a t i o n was o b s e r v e d with N31 (uvrB51) a n d N12-2 (uvrC53), w h e r e a s only a little a n d v i r t u a l l y n o r e a c t i v a t i o n t o o k p l a c e in N14-4 (uvrD3) a n d N611 (polA), respectively. D a t a confirming these results were o b t a i n e d with o t h e r strains having different m u t a t i o n s in these genes: AB1886 (uvrA6), AB1885 (uvrBS) a n d N13-1 (uvrD2). On the o t h e r h a n d , the results with uvrC m u t a n t s were s o m e w h a t c o n f u s i n g ; N12-2 (uvrC53) a n d AB1884 (uvrC34) r e s p o n d e d well to the enzyme, b u t N17-2 (uvrC55) s h o w e d a l m o s t no r e a c t i v a t i o n . Possible imp l i c a t i o n o f this o b s e r v a t i o n will be discussed later.

Discussion

a) Insertion of Enzyme into Permeable Cells In the present investigation we have d e m o n s t r a t e d t h a t a d e q u a t e l y p e r m e a b i l i z e d cells of E. coli m u t a n t s

44

K. Shimizu and M. Sekiguchi: Introduction of Enzyme into Permeable Cell

which are sensitive to u. v. acquire considerable degrees of u. v. resistance after treatment of the cells with T4 endonuclease V. Under appropriate conditions survivals of the enzyme-treated cells increased more than two hundred fold that of untreated cells. Detailed analyses of the phenomenon indicated that the following three conditions are required for the reactivation. (1) Cells must be permeabilized before treatment with T4 enzyme. Treatment of intact cells with enzyme did not cause any increase in survival after u. v. irradiation. (2) A specific action of T4 endonuclease V is necessary for the reactivation. The levels of u. v. resistance acquired were almost proportional to the amounts of the enzyme added to permeable cells. A pre-heated enzyme preparation, or enzyme fractions derived from cells infected with an endonuclease V-defective mutant of T4 exhibited only little or no effect on survivals of irradiated, permeable cells. (3) The recipient cells to be reactivated must be defective in uvrA, uvrB or uvrC gene. Other u. v.sensitive mutants, such as recA and polA mutants, were not reactivated, and u. v. resistance level of the wild-type strain was not affected by the enzyme addition. These characteristics of the reactivation are in conformity with the notion that T4 endonuclease V is taken up by permeable cells. The enzyme must have an access to the sites of action, namely u. v.-damaged sites on the chromosomal DNA, to exert such effect on irradiated cells. Furthermore, to form a visible colony, the enzyme-treated cell must undergo at least 20 cycles of cell divisions. Thus, the observed phenomenon can be taken as a strong proof that an active enzyme is inserted into viable cells. It has been shown that T4 endonuclease V introduces a single-stranded break at the 5'-side of a pyrimidine dimer in DNA, thereby initiating excision repair reactions in T4-infected cells (Yasuda and Sekiguchi, 1970b). Accumulating evidences suggest that the uvr genes of E. coli control a similar incision step (Howard-Flanders etal., 1966; Braun and Grossman, 1974), whereas thepolA gene product, D N A polymerase I, is involved in the latter step of excision repair and the recA gene controls other pathways for repair (De Lucia and Cairns, 1969; Boyle et al., 1970; Dorson and Moses, 1978; Howard-Flanders et al., 1969; Smith and Meun, 1970). These results are consistent with the present finding that permeable cells of uvrA and uvrB mutants, but not o f p o l A and recA mutants, acquired u. v. resistance on introduction of T4 endonuclease V. It seems, therefore, that the defective uvr functions of E. coli are complemented by the action of exogenously supplied T4 enzyme.

To calculate the number of dimers repaired by T4 endonuclease V, we adopted the vR sector, which was originally introduced by Harm (1968). As shown in the Results section, the maximal vR sector for the uvrA recA mutants was 34% at the u. v. dose giving 10.6 lethal hits when 25 units/ml of T4 enzyme was applied. Since 1.3 dimers per genome yields one lethal hit for the mutants (Howard-Flanders et al., 1969), it can be estimated that of average 14 dimers per genome present in the mutant about 5 dimers were repaired by the inserted enzyme. Similarly, the vR sectors for uvrA and uvrB mutants were calculated to be 46% and 39%, respectively, at the same enzyme concentration. Harm (1968) demonstrated that the u. v. survival of uvr mutants can be considerably increased upon infection with heavily u. v.-irradiated T4D, but not with similarly irradiated T4v mutant, and estimated the efficiency of the v gene action for repair of E. coli D N A in a cell. The maximum vR for a uvrA recA mutant and a uvrA mutant, calculated from his data after normalization for the host killing activity of phage, were 40% and 33%, respectively. Since these values are roughly equivalent to those obtained for the corresponding mutants in the present study, it is suggested that a considerable amount of T4 enzyme, comparable to that produced in T4-infected cells, is indeed inserted into E. coli cells. Of several methods which are known to increase permeability of cells, plasmolysis with high concentration of sucrose in the presence of E G T A was found to be most effective for the reactivation. Plasmolysed cells have been shown to be permeable to nucleotides and other compounds, and have been used for studies on mechanisms of D N A synthesis and other cellular processes (Wickner and Hurwitz, 1972; Wovcha et al., 1973; Seeberg and Strike, 1976; Stafford et al., 1977). Furthermore, Wickner and Hurwitz (1972) demonstrated that plasmolysed cells were permeable to some protein molecules, such as pancreatic DNase I and trypsin, although their plasmolysed cell preparations were practically non-viable (less than 0.1% of original viability). In the present study, by controlling various factors and conditions for plasmolysis, we were able to prepare permeable cells still retaining the colony-forming ability. Since the integrity of cellular organization is conserved in such viable permeable cells, this procedure may allow to investigate complex biological reactions in close association with organized cell structures. In addition, this system should provide useful means for identifying and assaying for proteins whose activity cannot be measured by conventional biochemical techniques. By using modifications of the present system, it might be possible to assay regula-

K. Shimizu and M. Sekiguchi: Introduction of Enzymeinto Permeable Cell tory proteins for cell division or proteins which are involved in processes of induction and fixation of mutation. There are, of course, some limitations in this system. The following problems must be considered on application of this system. (1) Foreign protein molecules inserted into cell may be degraded rapidly. Even if the protein is stable, it is diluted out through cell divisions. Thus, the consequences of a transient reaction, carried out by inserted proteins, must be fixed and enlarged through other cellular processes or cell reproduction. (2) Larger protein molecules might have greater difficulty in penetrating through cell membrane. Conditions for permeabilization of cells must be re-examined for each protein species. (3) It is inevitable to introduce other compounds together with an enzyme into cell. Appropriate controls are essential to exclude the possibility that the observed biological effects are caused by contaminating substances.

b) In situ Cornplementation of a uvr Function

Howard-Flanders and coworkers (1966)have demonstrated that uvrA, uvrB and uvrC mutants of E. coli are all defective in excising in vivo u. v.-induced pyrimidine dimers from DNA. Since the process of excision of dimers appears to consist of at least two reactions, introduction of a strand break near a dimer (incision step) and release of dimer-containing nucleotides from the incised region (excision step) (Yasuda and Sekiguchi, 1970b), it was supposed that each one of the uvr genes may control either or both of these steps. Although involvement of the uvrA and uvrB gene products in the incision step has well been established (Shimada et al., 1968; Seeberg and Johansen, 1973), there has been some controversy as to the role of uvrC gene. Kato (1972) demonstrated that u. v.induced incision took place in vivo in uvrC mutants; however, the rate of incision in uvrC mutants was far less than that of the wild-type strain (Seeberg and Johansen, 1973). It was shown, moreover, that a uvrC mutant possesses an endonuclease activity specific for u.v.-irradiated DNA, whereas uvrA and uvrB mutants do not (Braun and Grossman, 1974). On the other hand, recent studies indicated that the uvrC gene product as well as the products of uvrA and uvrB genes are required for ATP-dependent incision of u. v.-irradiated D N A in both plasmolysed cells and in a cell-free system (Seeberg et al., 1976; Seeberg, 1978). These situations prompted us to investigate this problem with the technique of in situ complementa-

45

tion. In this paper we presented evidence that impaired repair ability of uvrC mutants can be rescued by a supply of T4-coded incision enzyme; the levels of u.v. resistance acquired by certain uvrC mutants were almost the same as those acquired by uvrA and uvrB mutants. This result seems to support the notion that the uvrC gene product is also involved in the incision step. Although some uvrC mutants responded only slightly to the enzyme, such an allelespecific response might be due to the regulatory nature of the gene product. We found further that in situ complementation with T4 endonuclease V takes place in the uvrD mutants though the extents of the reactivation are very low. It has been shown that a uvrD mutation is dominant over the wild type allele and causes post-irradiation D N A degradation (Ogawa et al., 1968; Yamamoto, 1976). Although the precise function of the uvrD gene product is obscure, it is likely that it acts as a coordinator for the incision reaction in vivo. If so, the block may be circumvented, only in part, by the action of the T4 enzyme. These results are reminiscent of the previous finding that pretreatment of u. v.-irradiated doublestranded phage D N A by T4 endonuclease V leads to a marked increase in infectivity when the activity is assayed on uvr mutants but not on wild type strain or rec mutants (Taketo et al., 1972). The implications drawn from the two types of experiments are essentially the same; namely, the incision step in E. coli is controlled by at least four genes, uvrA, uvrB, uvrC and uvrD. However, there are some significant differences between the two systems. First, the present approach is more direct than is the previous one. In the previous experiment it was difficult to exclude a possibility that viral D N A incised in vitro may be converted, during infection, to other form(s) by an enzyme which is present in periplasm and is not normally involved in the repair reactions; this raises a question as to whether some gene product might act in such a way as to convert the incised D N A to some other intermediate (e.g. dephosphorylated DNA). In the present experiment the enyzme was inserted into cell to function in situ, and thus such a possibility is remote. The second major difference between the two systems is the amounts of the enzyme required for the reactivations. More than 20 units/ml of T4 enzyme was needed to obtain 0.5 vR sector in the permeable cell system whereas as low as 0.1 unit/ml of the enzyme was sufficient to produce the maximum reactivation for irradiated phage D N A (K. Oeda, unpublished results). It is supposed that only a small fraction of T4 enzyme may be incorporated into the cell, and, even though incorporated, may have difficulty to reach damaged sites on the DNA.

46

K. Shimizu and M. Sekiguchi : Introduction of Enzyme into Permeable Cell

T h e i n v o l v e m e n t o f at least f o u r genes in c o n t r o l ling t h e i n c i s i o n step, t o g e t h e r w i t h t h e f a c t t h a t t h e r e is r e l a t i v e l y a s m a l l a m o u n t o f i n c i s i o n e n z y m e a c t i v ity in E . coli, s t r o n g l y s u g g e s t s t h a t t h e p r o c e s s in E . c o l i is m o r e c o m p l e x t h a n t h a t f o u n d in p h a g e T4, in w h i c h o n l y o n e g e n e is i n v o l v e d in t h e i n c i s i o n step. T h e s e are s o m e p o s s i b l e e x p l a n a t i o n s o f t h e functions of the uvr genes; an active form of the incision enzyme may be composed of more than two protein subunits, or some other protein factor(s) besides an e n z y m e m a y be r e q u i r e d f o r l o c a l i z a t i o n a n d transportation of the enzyme on the chromosome. T h e t w o a p p r o a c h e s , o n e d e s c r i b e d b y T a k e t o et al. (1972) a n d a n o t h e r r e p o r t e d h e r e , m a y be useful f o r resolution of these problems.

We thank Drs. Y. Okada and T. Horiuchi for discussion and Mr. K. Yamashita for providing some preparations of T4 endonuclease V. This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan.

References Boyle, J.M., Paterson, M.C., Setlow, R.B.: Excision-repair properties of an Escherichia coli mutant deficient in DNA polymerase. Nature 226, 708-710 (1970) Braun, A., Grossman, L.: An endonuclease from Escheriehia coli that acts preferentially on UV-irradiated DNA and is absent from the uvrA and uvrB mutants. Proc. Natl. Acad. Sci. USA. 71, 1838 1842 (1974) Buttin, G., Kornberg, A. : Enzymatic synthesis of deoxyribonucleic acid. XXI. Utilization of deoxyribonucleoside triphosphates by Escherichia coli cells. J. Biol. Chem. 241, 5419 5427 (1966) Clark, A.J., Margulies, A.D.: Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc. Natl. Acad. Sci. USA. 53, 451-459 (1965) De Lucia, P., Cairns, J.: Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224, 1164-1166 (1969) Dorson, J,W., Moses, R.E. : DNA polymerase I-mediated ultraviolet repair synthesis in toluene- treated Escherichia coli. J. Biol. Chem. 253, 665 670 (1978) Dfirwald, H., Hoffmann-Berling, H. : Endonuclease I-deficient and ribonuclease I-deficient Escherichia eoli mutants. J. Mol. Biol. 34, 331 346 (1968) Furusawa, M., Nishimura, T., Yamaizumi, M., Okada, Y. : Injection of foreign substances into single cells by cell fusion. Nature 249, 449 450 (1974) Harm, W. : Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19, 66-71 (1963) Harm, W. : Recovery of UV-inactivated E. coli cells by the v-gene action of phage T4. Mutat. Res. 6, 175-179 (1968) Howard-Flanders, P., Boyce, R.P., Theriot, L. : Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53, 1119-1136 (1966) Howard-Flanders, P., Theriot, L., Stedeford, J.B. : Some properties of excision-defective recombination-deficient mutants of Escherichia coli K-12. J. Bact. 97, 1134--1141 (1969)

Kato, T. : Excision repair characteristics of recB- res- and uvrC strains of Escherichia coll. J. Bact. 112, 1237-1246 (1972) Lederberg, J., Clair, J. ST.: Protoplasts and L-type growth of Escherichia coll. J. Bact. 75, 143-160 (1958) Leive, L. : A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl, Acad. Sci. USA. 53, 745-750 (1965) Low, B.: Formation of merodiploids in mating with a class of Rec- recipient strains ofEscherichia coli K12. Proc. Natl. Acad. Sci. USA. 60, 160-167 (1968) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275 (1951) Loyter, A., Zakai, N., Kulka, R.G.: "Ultramicroinjection" of macromolecules or small particles into animal cells: A new technique based on virus-induced cell fusion. J. Cell Biol. 66, 292-304 (I975) Mandel, M., Higa, A.: Caicium-dependent bacteriophage DNA infection, J. Mol. Biol. 53, 159 162 (1970) Miura, A., Tomizawa, J.: Studies on radiation-sensitive mutants of E. coli III. Participation of the Rec system in induction of mutation by ultraviolet irradiation. Mol. Gen. Genet. 103, 1 10 (1968) Moses, R.E. : Replicative deoxyribonucleic acid synthesis in a system diffusible for macromolecules. J. Biol. Chem. 247, 6031~5038 (1972) Moses, R.E., Richardson, C.C.: Replication and repair of DNA in cells of Escherichia coli treated with toluene, Proc. Natl. Acad. Sci. USA. 67, 674-681 (1970) Ogawa, H.: Genetic locations of uvrD and pol genes of E. coli. Mol. Gen. Genet. 108, 378 381 (1970) Ogawa, H., Shimada, K., Tomizawa, J.: Studies on radiationsensitive mutants of E. coli I. Mutants defective in the repair synthesis. Mol. Gem Genet. 101, 227-244 (1968) Otsuji, N., Higashi, T., Kawamata, J. : Genetic and physiological analysis of mitomycin C-sensitive mutants of Escherichia coli K12. Biken J. 15, 49 59 (1972) Papahadjopoulos, D., Poste, G., Mayhew, E.: Cellular uptake of cyclic AMP captured within phospholipid vesicles and effect on cell-growth behaviour. Biochim. Biophys. Acta. 363, 404-418 (1974) Sato, K., Sekiguchi, M. : Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: The structural gene for T4 endonuclease V. J. Mol. Biol. 102, 15-26 (1976) Seeberg, E.: Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA + and uvrB+/uvrC + gene products. Proc. Natl. Acad. Sci. USA. 75, 2569-2573 (1978) Seeberg, E., Johansen, I. : Incisions in ultraviolet irradiated circular bacteriophage 2 DNA molecules in excision proficient and deficient lysogens ofE. coli. Mol. Gen. Genet. 123, 173 184 (1973) Seeberg, E., Nissen-Meyer, J., Strike, P.: Incision of ultravioletirradiated DNA by extracts of E. coli requires three different gene products. Nature 263, 524 526 (1976) Seeberg, E., Strike, P.: Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli. J. Bacteriol. 125, 787 795 (1976) Sekiguchi, M., Shimizu, K., Sato, K., Yasuda, S., Ohshima, S.: Enzymic mechanism of excision-repair in T4-infected cells. In: Molecular mechanisms for repair of DNA (ed. by P.C. Hanawalt and R.B. Setlow), Part A, pp. 135-142. New York: Plenum Press 1975 Shimada, K., Ogawa, H., Tomizawa, J.: Studies on radiationsensitive mutants orE. coli II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda. Mol. Gen. Genet. 101,245-256 (1968) Shimizu, K., Sekiguchi, M.: 5 ' ~ 3 ' Exonucieases of bacteriophage T4. J. Biol. Chem. 251, 2613-2619 (1976)

K. Shimizu and M. Sekiguchi: Introduction of Enzyme into Permeable Cell Shimizu, K., Sekiguchi, M. : Insertion of a functional repair enzyme into viable cells of Escherichia coli. Proceedings of the 1977 molecular biology meeting of Japan, pp. 93-95 (1977) Smith, K.C., Meun, D.H.C. : Repair of radiation-induced damage in Escherickia coli I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51,459-472 (1970) Stafford, M.E., Red@, G.P.V., Mathews, C.K.: Further studies on bacteriophage T4 DNA synthesis in sucrose-plasmolyzed cells. J. Virol. 23, 53 60 (1977) Taketo, A., Yasuda, S., Sekiguchi, M. : Initial step of excision repair in Escherichia coli: Replacement of defective function of uvr mutants by T4 endonuclease. V. J. Mol. Biol. 70, 1-14 (1972) Tanaka, K., Hayakawa, H., Sekiguchi, M., Okada, Y.: Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo. Proc. Natl. Acad. Sci. USA. 74, 2958-2962 (1977) Tanaka, K., Sekiguchi, M., Okada, Y. : Restoration of ultravioletinduced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendal virus). Proc. Natl. Acad. Sci. USA. 72, 4071 4075 (1975) Vosberg, H-P., Hoffmann-Berling, H.: DNA synthesis in nucleotide-permeable Escherichia coli cells I. Preparation and properties of ether-treated cells. J. Mol. Biol. 58, 739 753 (1971)

47

Wickner, R.B., Hurwitz, J.: DNA replication in Escherichia coli made permeable by treatment with high sucrose. Biochem. Biophys. Res. Commun. 47, 202 211 (1972) Wovcha, M.G., Tomich, P.K., Chiu, C., Greenberg, G.R. : Direct participation of dCMP hydroxymethylase in synthesis of bacteriophage T4 DNA. Proc. Natl. Acad. Sci. USA. 70, 2196-2200 (1973) Yamamoto, Y. : Studies on DNA degradation after UV-irradiation in uvrD mutant of Escherickia coll. Proceedings of the 1976 molecular biology meeting of Japan, pp. 136-137 (1976) Yasuda, S., Sekiguchi, M. : Mechanism of repair of DNA in bacteriophage II. Inability of ultraviolet-sensitive strains of bacteriophage in inducing an enzyme activity to excise pyrimidine dimers. J. Mol. Biol. 47, 243-255 (1970a) Yasuda, S., Sekiguchi, M.: T4 endonuclease involved in repair of DNA. Proc. Natl. Acad. Sci. USA. 67, 1839-1845 (1970b) Yasuda, S., Sekiguchi, M.: Further purification and characterization of T4 endonuclease. V. Biochim. Biophys. Acta 442, 197-207. (1976) Zinder, N.D., Arndt, W.F. : Production of protoplasts of Escherickia coli by lysozyme treatment. Proc. Natl. Acad. Sci. USA. 42, 586-590 (1956) C o m m u n i c a t e d b y T. Y u r a Received August 26, 1978