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Initial step of excision repair in Escherichia coli: Replacement of defective function of uvr mutants by T4 endonuclease V
J. Mol. Biol. (1972) 70, 1-14
Initial Step of Excision Repair in Escherichiu cd: Replacement of Defective Function of uvr Mutants by T4 Endonuclease V AKIRA
TAKETO?,
SEIICHI
YASUDA
AND MUTSUO
SEKIGUCHI
Department of Biology, Faculty of Science Kyu.shu University, Fukuoka, Japan (Received 24 April
1972)
The ultraviolet sensitivity of double-stranded replicativo form and singlestranded DNA of bacteriophage +A and +X174 was studied by using spheroplasts of various strains of Escherichiu co.%. The replicative form is more sensitive when assayed on uvr- mutants than when plated on wild type or ret- mutants, while t,he single-stranded DNA exhibits the same high sensitivity on these strains. Pretreatment of ultraviolet-irradiated RF$ by a purified preparation of T4 endonuclease V leads to a marked increase in biological activity of DNA when t,he activity is assayed on uvr- mutants but not on the wild type or ret- mutants; the same treatment, however, has no detectable effect on the activity of nonirradiated RF. Thus, the function in vivo controlled by the uvr genes of E. coli can be replaced by the reaction in vitro catalysed by T4 endonuclease V. It is suggested that the first step of excision repair in E. coli is to induce a singlestranded break at a point close to a pyrimidine dimer, possibly to the 5’ side of a dimer, in DNA, and that, the step is controlled by at, least four genes, UWA, uvrB, uvrC and uvrD.
Irradiated RF treated with T4 endonuclease V or pancreatic deoxyribonuclease, or non-irradiated RF treated with the pancreatic enzyme, exhibit lower infectivity when assayed on pal- strains as compared to assaying on pal+ strains. Treatment of irradiated RF by both T4 endonuclease V and excision enzyme, which produces gaps several niicleotides in length in the molecule, also decreased tho I biological act,ivity of RF in poZ- st,rains. It seams that E. co& DNA polymerase is required to repair gaps and nicks in DNA.
1. Introduction Mechanism of repair of DNA by excision has been studied extensively using mutants of Escherichia coli sensitive to ultraviolet radiation. It has been demonstrated that strains having mutation in one of uvrA, uvrB and uvrC genes are unable to excise U.V. induced pyrimidine dimers from DNA; thus, at least three different genes may control the excision process (Setlow & Carrier, 1964; Boyce & Howard-Flanders, 1964; Howard-Flanders, Boyce & Theriot, 1966). It has been shown, moreover, that strains with mutation in uvrD, polA or ret genes degrade their own DNA extensively after U.V. irradiation, suggesting that these genes are responsible for repair synthesis after 1968; De Lucia & Cairns, 1969; Boyle, excision (Ogawa, Shimada & Tomizawa, Paterson & Setlow, 1970; Kato & Kondo, 1970). t On leave from the Department of Biochemistry, Kanazawa University $ Abbreviation used: RF, double-stranded replicative form DNA. 1 1
Medical
School.
2
A. TAKETO,
S. YASUDA
AND
&I. SEKIGUCHI
Although great efforts have been made to elucidate the enzymic mechanism of excision repair in E. coli, no enzyme responsible for excision of pyrimidine dimers has been identified. An endonuclease activity that is specifically active on u.v.-irradiated DNA was found in an extract of E. coli; however, all the mutants so far examined. including those unable to excise dimers in viva, also possessed the enzyme activity (Takagi et al., 1968). We have shown that an extract of E. coli infected with bacteriophage T4 contains a high level of an enzyme activity that induces single-stranded breaks specifically in u.v.-irradiated DNA (Yasuda & Sekiguchi, 1970aJ; Friedberg & King, 1971). The enzyme, T4 endonuclease V, is not, found in an extract of cells infected with u.v.sensitive mutants of T4; thus, it seems probable that the enzyme is responsible for the first step of excision repair in T4-infected cells. A similar endonuclease activity has been found in Micrococcus Euteus (M. lysodeikticus) (Strauss, Searashi & Robbins. 1966; Takagi et aE., 1968; Grossman, Kaplan, Kushner & Mahler, 1968; Carrier & Setlow, 1970; Nakayama, Okubo & Takagi, 1971). The availability of such an endonuclease should make it possible to study the initial steps of excision repair in E. coli. If the first step of excision repair in E. coli is catnlysed by an enzyme similar to T4 endonuclease V, u.v.-irradiated infective viral DNA, once treated in vitro by the T4 enzyme, may be repaired by mutants defective in the incision step (Fig. 1). This paper describes the results of such experiments.
Wild type
-
+
+
+
Mutant I
-
-
+
+
Mutant Ii
-
Mutant IJI
-
-
-
+
-
-
FIG. 1. Schematic representation of the proposed method for the analysis of excision--repo.it mechanism of E. coli. Double-stranded DNA, pre-exposed to u.v., can be repaired by the excisioll+ repair system of E. coli, whereas single-stranded phage DNA cannot. E. coli mutants which at)‘~ defective in one of the steps in repair are unable to repair irradiated DNA. When, howevt,r, irradiated double-stranded DNA is converted to intermediate forms, such intermediates may 1)(x repaired by mutants which are defective in the earlier steps but not in the later ones. ( + ) Repairable; (-) non-repairable.
2. Materials and Methods (a) Bacteria
and bacteriophage
The UVT- mutants used are derived from two distantly related E. coli K12 strain);: N17-9 (u~~A54), N3-1 (uv~B51), N17-7 (uvrC56) and N14-4 (uvrD3) derived from W3623 (Ogawa et al., 1968); and AB1886 (uvrAG), AB1885 (uvrB5) and AB1884 (uurC34) derived
REPAIR
OF DNA
IN
E. COLZ
3
used, AB2463 (recAl3), from AB1157 (Howard-Flanders et aE., 1966). The ret- mutants AB2470 (recBZl), SR88 (recC22), JC5547 (recA13 recB21 recC22), are derivatives of AB1157 (Willetts & Clark, 1969; Smith & Meun, 1970). Double mutants, JC5412 (recB21 $5~8) (Barbour, Nagaishi, Templin & Clark, 1970) and N212 (recA1 uwrA54) (Miura & Tomizawa, 1968), were also used. DNA polymerase I-deficient mutants, P3478 (poEAl) and N611 (pal). are derived from W3110 and W3623, respectively (De Lucia & Cairns, 1969; Ogawa, 1970). Bacteriophage +A is a spherical virus containing single-stranded circular DNA and immunologically related to #X174 (Taketo t Kuno, manuscript in preparation). The phage was propagated in E. coli C and used in most of these experiments. Bacteriophage +X1 74 and T4 were also used in some of the experiments. (b) Enzymes T4 endonuclease V was prepared from T4-infected E. coli 1100 by a modification of the procedure previously described (Yasuda & Sekiguchi, 19705). The purification procedure, details of which will be published elsewhere, includes phase partition in dextran/polyothelene glycol and column chromatography of CM-Sephadex C-25 and of Hypatite C. The enzyme was purified about 1300-fold and is free of other endo- and exonucleases. E. coZi N29 (poZ- endoI- ), was provided by Excision enzyme, isolated from T4iv,-infected Mrs S. Ohshima of this laboratory. The enzyme excises dimer-containing nucleotides from u.v.-irradiated DNA, which has been treated with T4 endonuclease V (Ohshima & Sekiguchi, 1972). Purified bovine pancreatic deoxyribonuclease was obtained from Sigma Chemical CO.
(c) Preparation
of DNA
Infective single-stranded DNA was extracted from purified +A particles by phenol treatment and dialysed against 0.05 M-TriseHCl buffer, pH 8.0 (Taketo I% Kuno, 1969). The +A DNA is converted into double-stranded replicative form upon infection of the host, E. coli C (Taketo & Kuno, 1972). To prepare RF, +A-infected cells were incubated with 25 pg chloramphenicol/ml for 90 min at 37”C, collected and lysed by lysozyme/EDTA/ sodium dodecyl sulphate method. After centrifugation at 100,000 g, the lysate was shaken with 0.05 M-borate-saturated phenol and subjected to isopropanol precipitation. The host DNA was removed by repeated winding with a glass rod and RNA was digested by ribonuclease. After the second phenol treatment and isopropanol precipitation, the material was applied to a methylated albumin column. The RF eluted at 0.65 M-NaCl was collected and dialysed against 0.05 M-Tris.HCl buffer, pH 8.0. RF of 4X174 was also prepared by a similar procedure. Labelled RF was prepared from #A-infected E. coli C thy-, incubated with 2 pg 1hymine/ml. (0.2 &i/pg), as described above.
(d) Irradiation
of DNA
DNA was irradiated with a 15 W Toshiba germicidal most experiments, irradiation was for 25 set at a distance mate dose: 600 ergs/mm2). The dose rate was determined 11.v. inmnsity meter.
lamp at room temperature. In of 25 cm from the lamp (approxiby a Blak-ray J-255 short-wave
(e) Assay of infectivity Infectivity of DNA was assayed on lysozyme/EDTA spheroplasts. Cells were harvested from an exponentially growing culture in 50 ml. of broth, washed with 10 ml’ of 0.01 MTris.HCl (pH 7.5) and suspended in 5 ml. of 20% sucrose/O.1 M-Tris.HCl (pH 8.0) followed by an addition of lysozyme (80 pg/ml.). After 10 min at room temperature, (0.08 ml. of 0.05 M-EDTA (pH 7.5) was added and the mixture was allowed to stand for a further 10 min. The reaction was terminated by addition of 0.1 ml. of 1 M-MgSO, and 0.05 ml. of 1 M-Call, and the spheroplasts were collected by centrifugation at 4000 g foi 10 min in the cold and suspended in 1 ml. of chilled 20% sucrose broth. To determine infectivity of DNA, 0.1 ml. of the spheroplast suspension was added to a mixture of 0.1 ml.
4
A. TAKETO,
S. YARUDA
AND
hr. AEKIQUCHI
of DNA in 0.05 M-‘i’ris.HCl (pH 84) and O-1 ml. of 2UU/, sucrose broth. The mixture was incubated at 37°C for 12 min, diluted with chilled 20% sucrose broth and then plated, at. least in duplicate, with a culture of E. co& C in top agar, which consists of 20% sucrose:/ 0.5% agar/broth. Throughout the process care was taken to avoid photoreactivation. (f) Determination
of pyrimidine
dimers
DNA and the acid-soluble fractions were hydrolysed by heating acid at 100°C for 3 hr. The amount of thymine and thymine-containing mined by Dowex-1 column chromat,ography (Sekiguchi et al., 1970).
with 6 N-perchloric dimers was deter-
3. Results (a) Host cell reactivation
of irradiated
replicutive
fornz DNA
Figure 2 indicates the u.v.-sensitivity of single- and double-stranded DNA of $A assayed on spheroplasts of various E. coli strains. The RF is more sensitive when assayed on u.v.-sensitive mutants having mutation in one of the uvr genes than on the wild-type strain (Fig. 2(a)). This is in agreement with the results obtained with $X174 RF (Jansz, Pouwels & Van Rotterdam, 1963; Yarus & Sinsheimer, 1964). As shown in Figure 2(b), there is a small but consistent difference between u.v.-sensitivity of the RF assayed on pal + and on pal- strains as observed with $X174 (Klein & Niebch, 1971); irradiated RF exhibits lower survival in DNA polymerase-deficient mutants, P3478 and N611, isolated from the different parents. On the other hand, no difference in u.v.-sensitivity is observed when the RF is assayed either on ret + or on ret - strains (Fig. 2(c)), though the ret- mutants themselves are sensitive to WV. In spite of the great difference of u.v.-sensitivity of the RF, single-stranded DNA exhibits almost an equally high u.v.-sensitivity when assayed on spheroplasts of various strains. These results are consistent with the idea that the RF of $A can be reactivated by a host cell mechanism, in which the double-stranded nature of DNA is required for repair of u.v.-induced lesions by excision and resynthesis. It appears that, E. coli genes uvrA, uvrB, uvrC and uvrD as well as polA are involved in the process. whereas recA, recB and recC are not.
(b) Effect of T4 endonuclease
V on the reactivation
of replicative
form DNA
If excision repair proceeds in sequential manner and the host cell reactivationnegative mutants are defective in one of the steps, intermediates in the repair process should be reactivated by mutants which are defective in the earlier steps but not in the later ones (see Fig. 1). In order to test this possibility, experiments were performed using T4 endonuclease V as an enzyme to produce such an intermediate. It has been demonstrated that the enzyme induces a single-stranded break at a point close to a pyrimidine dimer (Yasuda & Sekiguchi, 1970b). In an experiment, the result of which is shown in Figure 3, +A RF was irradiated with various doses of U.V. and then incubated with or without T4 endonuclease V at, 37°C for 30 min in 0.04 M-Tris*HCl, pH 7.5. After appropriate dilution, the DNA’s were mixed with spheroplasts of wild-type E. coli W3623 and of its uvrA- mutant, N17-9, and infectivity was determined. As can be seen in the Figure, preincubation of irradiated RF with T4 endonuclease V leads to a marked increase of biological activity of the RF when the activity is assayed on the uvrA- mutant but not on the wild-type strain; the same treatment, however, has no detectable effect on the activity
’ +. \1’ \ \ A ‘1b\
(0)
lh
i
(b)
1..
10-I - “a\ \ A\A ‘1\ g \ \\ \0 8 A\ :\ lo+t ‘1\ \\ \ “\ \o .\ \\ 1O-3: \\ \ .-6 ‘?\ B I I I I -F lo-4 ‘5 .-> c (cl ‘;;\. \\ -----I--b \\ ‘4.
\
\
i‘, ‘? \
‘? /AL 5
IO
u v. Irradmtlon
15
20
time (set)
\* \
1
I
I
I
5
IO
15
20
uv. Irrodiatton tipe (set)
2. Host cell reactivation of $A RF. RF (3.8 x lo4 plaque-forming units/ml. on E. coli C spheroplast) or single-stranded DNA (3.8 x lo6 plaque-forming units/ml. on E. coli C spheroplast) of +A was irradiated at a distance of 25 om from a 15 W germicidal lamp (Toshiba) and the infectivity was assayed on spheroplasts of various E. coli strains. (a) uvr System: (0) W3623 (wild); (0) Nli’-9 (uwrA); (A) N3-1 (uvrB); ( q i) N17-7 (uurC); (A) N14-4 (uvrD). (b) ~oZ system: (0) W3623 (wild); (A) N611 (pal); (A) P3478 (poZA). (c) ret system: (0) W3623 (wild); (011 AB2463 (recA); (a) AB2470 (recB); (0) SR88 (recC). (-) RF; (----) single-stranded DNA.. FIG.
G
A. TAKETO,
S. YASUDA
,0-l 0
,:
AND
I
IO 20 u v. Irrodation
M. SEKIGUCHI
I
1
30 tune (SK)
40
on the infectivity of u.v.-irradiated RF. RF or FIG. 3. Effect of T4 endonucleaae V treatment single-stranded DNA of $A was irradiated, as described in Fig. 2, and then incubated with or without T4 endonuclease V at 37°C for 30 min in 0.04 M-Tris .HCl buffer, pH 7.5. After suitable dilution, the infectivity was assayed on W3623 (wild) or N17-9 (uurA) spheroplasts. ( 0) Untreated DNA, assayed on W3623; (A) untreated DNA, assayed on N17-9; (0) enzyme-treated DNA, assayed on W3623; (A) enzyme-treated DNA, assayed on N17-9. ((--) RF; (----) singlestranded DNA.
of non-irradiated RF. This indicates that the reaction in vitro catalysed by T4 endonuclease V replaces, to a certain extent, the function in viva controlled by the uvrA gene of E. co&. (c) Comparison of reactivation
capacities
of E. coli mutants
The abilities of various E. coli mutants to reactivate irradiated RF which has been treated with T4 endonuclease V were next compared. The results, summarized in Tables 1 and 2, indicate that uvrA-, uvrB- and uvrC- mutants respond strongly to the enzyme treatment; 15 to 75fold increase of the biological activity is observed with both the AB and N series of uvr- mutants, isolated from AB1157 and from W3623, respectively. A small but significant reactivation was observed with uvrDmutant. As expected, ret- mutants, which have normal capacity for reactivation, do not respond to the enzyme treatment. Of interest is the observation that pal- mutants exhibit lower survival when irradiated RF has been treated with the enzyme. Effect of T4 endonuclease V is specific for irradiated RF. When non-irradiated RF is incubated with the enzyme, no significant change in the biological activity is observed, irrespective of the types of mutants employed (Table 1). Moreover, it was shown that treatment of irradiated RF with pancreatic deoxyribonuclease has little effect on the activity assayed on various hosts (Table 2). The pancreatic enzyme is known to induce single-stranded breaks, having no specificity for pyrimidine dimers. This will be discussed later in more detail. The kinetics of the reaction are shown in Figure 4. In these experiments the RF was irradiated at the fixed dose (for 25 seconds at a distance of 25 cm from a lamp;
REPAIR
OF DNA
IN
7
E. COLI
TABLE 1 Effect of T4 endonucleu.se V treatment on infectivity of u.v.-irradiated or non&m&ted +A RF Relative infectivity after T4 endonuclease V treatmentt
Surviving fraction of irradiated RF
Strains
AB1157 (wild) AB1886 (uvrA) AB1885 (uurB) AB1884 (uwrC) JC5547 (recA recB recC) JC5412 (recB &A) N212 (recA uurA) P3478 (poZA)
Irradiated RF
2.2 x 10-l 2.1 x10-s 2.4~10-~ 3.8 x 1O-3 2.3 x10-i 2.1 x10-i 7.8~10-~ 8.7 x 1O-2
1.00 15.7 75.0 29.7 1.06 I.02 36.7 0.41
Non-irradiated RF 0.93 0.96 0.99 I.05 1.10 1.00 1.16 1.00
,$A RF was irradiated et a disttmce of 25 cm from the lamp for 25 set and divided into two portions. One portion was incubated with T4 endonucle~e V (0.4 pg protein/ml.) in 0.05 M-Tris. HCl (pH 7.5) et 37°C for 10 mm, 8nd the other was incubated without the enzyme. Non-irradiated RF was also divided into two portions and treated in the same manner. The four types of sample were subjected to infectivity assay using spheroplasts of various E. coli strains. + Ratio
of T4 endonuclease
V-treated
RF to untreated
RF.
TABLET Effect of treatment with T4 endonuclease V or pancreatic deoxyribonuclease on infectivity of irradiated $A RF
Strains
W3623 (wild) N17-9 (uurA) N3-1 (uvrB) N17-7 (WC) NlP4 (uurD)
Surviving fraction of irradiated RF 1.0x 2.0x 2.3 x 1.3 x 1.7 x
10-i 10-3 1O-3 10-s IO-2
Relative
infectivity of irradiated RFt after treatment with Pancreatic T4 endonuclease V DNase 0.90 34.6 43.7 40.1 3.63
0.89 0.67 1.00 0.75 0.80
4A RF was irradiated with U.V. at B distrmce of 25 cm for 25 set and divided into three portions: (1) treated with T4 endonuclease V (0.4 pg protein/ml.) in 0.05 M-Tris.HCl (pH 7.5) at 37’C for 10 min; (2) treated with pancreatic DNase (0.001 pg/ml.) in 0.05 M-Tris .HCl/O.Ol nr-MgSo, (pH 7.5‘) at 37°C for 10 min; (3) without treatment. The three types of samples were subjected to infectivity assay using spheroplasts of various E. coli strains. t Ratio of enzyme-treated RF to untreated RF.
approximately 600 ergs/mm2) and treated with T4 endonuclease V at different concentrations for 10 minutes (Fig. 4(a) ) or at a concentration of O-4 pg protein/ml. for different times (Fig. 4(b)). The reaction was terminated by shaking with phenol and the DNA, recovered from the aqueous layer and dialysed, was assayed on spheroplasts of various strains. The mutants examined were uvrA-, uvrB- and uvrC-> derived from AB1157, and P3478 (~oZA) (Fig. 4(a)), and uvrA-, uvrB-, uvrC-, uvrD--
8
A.
TAKETO,
1 01
1o-3 ’ 0
I. 02
Concentration
S. YASUDA
I-/: 03
04
AND
05
0
of enzyme (~9 protein/ml
)
M.
SEKIGUCHI
5
I IO
I 15
I 20
Ttme of mcubatlon(min)
FIG. 4. Kinetics of the reaction. (a) Effect of concentration of T4 endonuclease V. RF, irradiatecl was incubated with various amounts of T4 endonuclease V at 37°C for 10 min at 600 ergs/md, in 0.05 iv-Tris.HCl, pH 7.5. After shaking with phenol, the DNA was dialysed and assayed on spheroplasts of the following strains. (0) AB1157 (wild); (0) AB1886 (uwTA); (A) AB188R (uvrB); (0) AB1884 (u&Z); ( n ) P3478 (poZA). (b) Time-course of the reaction. RF, irradiated at 600 ergs/mm2, was incubated with 0.4 pg protein/ml. of T4 endonuclease V at 37°C in 0.04 x-T&. HCl, pH 7.5. At the times indicated, the reaction was terminated by shaking with phenol and thP biological activity was assayed, after dialysis, on spheroplasts. (0) W3623 (wild); (0) N17-9 (uvrA); (A) N3-1 (uvrB); (0) N17-7 (uvrC); (A) N14-4 (uvrD); ( n ) N611 (pal).
and pal-, derived from W3623 (Fig. 4(b)). F rom the results, it is evident that t’he reactions are functions of both time and enzyme concentration. With increasing time of incubation or concentration of enzyme the activities assayed on uvr- mutants increased, while those assayed on pal- mutants decreased. At the plateau, some uvrmutants exhibit almost the same high level of reactivation as do the wild-type strains. It is worthy of note that uvrD- mutant is able to reactivate the enzyme-treated RF. t,hough the mutant differs from other uvr- mutants in many properties (Ogawa et al., I 968). TABLE
Effect of T4 endonuclease
1’ treatment or non-irradiated
W3623 (wild) Nl7-9 (uvrA) N3-1 (uvrB) N17-7 (WC) N14-4 (uvrD)
on infectivity $X174 RF
of u.v.-irradiated
Relative infectivity after T4 endonuclease V treatmenti
Surviving fraction of irradiated RF
Strains
3
Irradiated RF
2.0 x 10-l 3.0 x 10-S 3.4x10-3 3.2x 1O-3 2.8 x 1O-2
1.8 24.3 54.5 17.5 4.5
Non-irradiated RF 0.93 0.78 0.83 0.87 I.08
The conditions and procedures were the same as described in the legend to Table 1 except +X174 RF was treated with a higher concentration (12 pg protein/ml.) of T4 endonuclease t Ratio
of enzyme-treated
RF
to unt,reated
RF,
that V.
REPAIR
OF DNA
IN
E. COLI
Similar results were obtained with RF of $X174; a considerable place when irradiated +X174 RF were treated with T4 endonuclease one of the four types of uvr- mutants (Table 3). (d) Infectivity
9 reactivation took V and assayed on
of RF with nicks or gaps
The foregoing experiments demonstrated that pre-treatment of irradiated RF b;y T4 endonuclease V results in a decrease of biological activity of DNA when the activit,y was assayed on pal- mutants. Since no such decrease was observed when the assa,y was made on the wild-type strain, it was suspected that nicks produced in RF by the endonuclease are more injurious in pal- cells than in pal+ cells. In order to test this possibility, we have investigated the effect of pancreatic deoxyribonuclease on the infectivity of RF in W3623 (poZ+) and N611 (pal-). As shown in Figure 5, the rate of I
Time of mcubation (min)
FIG. 5. Inactivation of u.v.-irradiated and non-irradiated RF by pancreatic deoxyribonuclease. +A RF, irradiated with u.v. (at 600 ergs/mm2), or non-irradiated RF, was incubated at 37°C wii,h 0.005 pg/ml. of pancreatic deoxyribonuclease in 50 mM-Tris*HCl/lO mM-MgSO,, pH 7.5. Samples were withdrawn at the times indicated and infectivity was assayed on spheroplasts of W3623 and RF on N611; ( 0) irradiated RF on N611. (0) Non-irradiated RF on W3623; (A) non-irradiated W3623; (A) irradiated RF on N611.
inactivation of both irradiated and non-irradiated RF is higher in the pal- strain than in the pal+ strain. It was shown, moreover, that irradiated RF is inactivated by thr enzyme more rapidly than is non-irradiated RF, probably due to introduction of additional nicks into irradiated RF by certain endonucleases specific for irradiated DNA in E. coli. These results are consistent with the idea that DNA polymerase I is involved in repair of nicks produced in RF by the enzyme treatments. Recently, it was shown that the combined action of T4 endonuclease V and excision enzyme results in the excision of dimer-containing nucleotides from u.v.irradiated T4 DNA (Ohshima & Sekiguchi, 1972). The result shown in Table 4 indicates that irradiated +A RF is also subjected to the action of these enzymes. On incubation with T4 endonuclease V or excision enzyme alone, no appreciable
45
0 1 1
(cts/min)
Acid-soluble
38
0 0.8 0.7
(Oh release)
Dimers
39
0 15 3
(cts/min)
fraction
2.5
0 0.9 0.2
(% release)
Thymine
73
112 126 142
(cts/min)
Dimers
0.75 0.77 0.77 0.47
15,350
dimers (%)
Content
14,900 16,150 17,900
(ctsjmin)
DNA fraction Thymine
of
[‘*C]Thymine-labelled 4A RF was irradiated at 600 ergs/mm2 and incubated at 37°C for 60 min. The reaction mixture (0.3 ml.) contained 0.2 pg of DNA, 12 pmoles of Tris-maleate (pH 7.0), 3 pmoles of MgCl, and T4 endonuolease V (0.2 pg) and/or excision enzyme (1.05 pg). After incubation, the mixture was acidified and separated into DNA and acid-soluble fraction. Each sample was hydrolysed by heating with 6 rr-perchloric acid at 100°C for 3 hr and the radioactivity of thymine and its dimers was measured after separation by Dowex 1 column chromatography.
None Endonuclease V Excision enzyme Endonuclease V plus excision enzymes
Enzyme
4
Effect of T4 endonuclease V and excision enzyme on the release of pyrimidine dimers from u.v.-irradiated +A RF
TABLE
REPAIR
OF DNA
IN
E. COLI
11
amount of dimers or thymine is released from the DNA. When, however, both enzymes are present in the reaction mixture, selective release of dimers takes place; about 40% of the dimers originally present in the DNA become acid-soluble, while only 3% of the thymine is released from the DNA. From the ratio of radioactivity in thymine and its dimers in the acid-soluble fraction and the thymine content of DNA, it is estimated that about six nucleotides are excised for each dimer. Thus, the RF treated by the two enzymes would possess gaps of several nucleotides in length in the molecule. Infectivity of RF treated with these enzymes was next investigated. As shown in Table 5, irradiated replicative forms, treated with T4 endonuclease V alone or with TABLE
5
Effect of T4 endonucleme V and excision enzyme on the infectivity of u.v.-irradiated q5A RP Infectivity W3623
Enzyme
None Endonucleese V Excision enzyme Endonuclease V plus excision enzyme QA RF w&s irradiated at 600 (0.3 ml.) contained irradiated endonucleaae V (3.7 pg) and/or W3623 or N611, w&s expressed
assctyed on N611
22.7 49-o 33.2
14.4 2.1 17.1
49.4
0.9
ergs/mn? snd incubated at 37’C for 10 min. The incubation mixture RF, 15 pmoles of Tris-HCl (pH 7.5), 3 pmoles of MgSO, and T4 excision enzyme (1.72 pg). Infectivity, sswyed on spheroplasts of 88 y. of non-irradiated RF (without enzyme treatment).
both endonuclease V and excision enzyme, exhibit almost the same low infectivity for pal- mutant. No such decrease of infectivity was observed when the treated RF was assayed on poZ+ strain. Thus, gaps as well as nicks in the DNA appear to be equally injurious in pal- cells.
4. Discussion Endonucleases which act specifically on DNA pre-exposed to U.V. radiation have been found in several micro-organisms. First, the activities were detected in extracts of M. lutew and also of E. coli (Strauss et al., 1966; Grossman et al., 1968; Takagi et al., 1968). The enzyme was purified from M. luteus and its properties have been studied extensively (Carrier & Setlow, 1970; Kaplan, Kushner & Grossman, 1971; Nakayama, Okubo & Takagi, 1971). Although the endonucleases have many characteristics expected of an enzyme that catalyses the first step of excision repair, the role of the enzymes in in vivo repair has not fully been understood. A number of u.v.-sensitive strains from E. coli and from M. 1uteu.s possess the normal level of endonuclease activities, while mutants lacking the enzyme are only slightly u.v.-sensitive (Okubo, Nakayama, Sekiguchi & Takagi, 1967; Takagi et al., 1968; Okubo, Nakayama cc5 Takagi, 1971; Mahler, Kushner t Grossman, 1971).
12
A. TLKETO,
S. YASUDA
ANI)
11. SEKIGT:(‘HT
Recently, it was found that T4 endonuclease I#*, which is similar to X. luteu.~ endonuclease in many respects, is induced in E. coli after infection with T4 (Yasuda $ Sekiguchi, 1970a$; Friedberg & King, 1971). The formation of the enzyme is controlled by the v+ gene of T4, the gene which is known to increase survival of phage after U.V. irradiation (Harm, 1963). It was shown, moreover, that excision of pyrimidine dimers in viva as well as in vitro takes place in T4v+ -infected cells but not in T4v--infected cells (Setlow & Carrier, 1966; Sekiguchi et al., 1970; Katsuki &Sekiguchi, manuscript in preparation), Thus, it seems probable that the enzyme is responsible for the first step of excision repair in T4-infected cells. In the present investigation, we have demonstrated that pre-treatment of u.v.irradiated double-stranded viral DNA by a purified preparation of T4 endonuclease \ leads to a marked increase of biological activity of DNA when the infectivity was assayed on E. eoli strains having mutation in the uvr,4, B, C or D gene. Since these mutants are unable to reactivate irradiated RF without the enzyme treatment, it ia suggested that T4 endonuclease V supplies a function that is missing in tbtb uvr mutants. It seems probable, therefore, that the first step of excision repair in E. coli is catalysed by an enzyme whose mode of action is similar to that of T4 undonuclease V. There is some evidence that uvrA, B and C genes control some step of the excision process. It has been demonstrated that strains with mutation in one of these genes are unable to excise pyrimidine dimers (Setlow & Carrier, 1964; Boyce 8~ HowardFlanders, 1964; Howard-Flanders et aE., 1966). It has been shown, moreover, that fragmentation of cellular DNA after u.v.-irradiation is less in these mutants than in the wild-type strain (Shimada, Ogawa & Tomizawa, 1968). The present finding that irradiated RF, once treated with T4 endonuclease V in vitro, can be reactivated b> uvr- mutants is in accord with these observations. We had not, however, expected reactivation of the enzyme-treated RF also to take place in the uvrD- mutant. Since the mutant exhibits an extensive degradation of DNA after u-v.-irradiation, it n-as suspected that the mutant is defective in the second or the later steps of excision repair (Shimada et al., 1968). This implies that the uvrD gene function is different. from the functions of the uvrA, B and C genes, even though all these genes may control the incision step. The involvement of at least four genes in controlling the incision step, together with the fact that there is no direct correlation between the u.v.-sensitivity and the in vitro enzyme activity in E. coli, strongly suggests that the process of excision repair in normal E. coli is more complex than that found in T4-infected cells. It is supposed that the repair in infected cells is carried out in the cytoplasm, where the efficiency of the reaction mostly depends on the amount and activity of enzymes involved. If we assume that the repair in normal cells takes place in some particular part of the cell, e.g. cell membrane, it is easily understood that other factors affecting localization and transportation of enzyme and substrate are much more important (Boyle & Setlow, 1970). It has been demonstrated that T4 endonuclease V introduces a break at the 5’ side of a pyrimidine dimer in irradiated DNA (Yasuda & Sekiguchi, 1970b). Thus, if t,he first step of dimer excision in E. coli is catalysed by an enzyme similar to T4 endonuclease V, then the enzyme that is responsible for the second step should be an exonuclease that hydrolyses a polynucleotide in a 5’ + 3’ direction, or else a specific endonuclease that induces a break to the 3’ side of a dimer (Fig. 6). It was demon-
3’
REPAIR
OF DNA
Endonucleose
V
IZ. COLI
5’
P
z
i 5’ 3’ FIG.
IN
6. A possible scheme for the initial
a Exonucleose b-3’) or Endonucleose
I4
5’
3’
steps in the repair of u.v.-irradiated
DNA.
strated that DNA polymerase I of E. coli possessesa 5’ + 3’ exonuclease activity and can excise dimer-containing nucleotides from irradiated DNA in vitro (Kelly, Atkinson, Huberman & Kornberg, 1969). It might be possible that both the second step of excision and repair replication are catalysed by a single enzyme, DNA polymerase I. However, no direct evidence has been obtained to indicate that DNA polymerase 1 is involved in excision of dimers in vivo. The rate and final extent of dimer excision are almost the same in the pal+ and POE- strains (Boyle et al., 1970; Katsuki & Sekiguchi, manuscript in preparation). Thus, there is a possibility that other enzyme(s) function t,o excise dimers or replace DNA polymerase I in the process. The present finding that the u.v.-sensitivity of #A RF inpol- mutants is low compared to that in uvr- mutants also supports this view. Recently, enzymes that catalyse the selective release of dimers from irradiated DNA, pre-treated with endonucleases, were isolated from M. luteus and T4-infected E. coli (Kushner, Kaplan, Ono & Grossman, 1971; Ohshima & Sekiguchi, 1972). It is evident that, apart from functioning in the excision of dimers, DNA polymerase I plays some role in repair of DNA. It has been shown in the present paper that RF possessing nicks or gaps exhibits lower survival in pal- mutants than in the wildtype strain. This implies that DNA polymerase I may be required for filling gaps and sealing nicks in DNA. Recently, it was demonstrated that joining of newly replicated DNA chains in viva is slow in pal- mutants (Kuempel & Veomett, 1970; Okazaki, Arisawa & Sugino, 1971). Finally, it is pointed out that the procedure employed in the present investigation is potentially useful for a specific assay of endonuclease that acts on irradiated DNA. It was demonstrated that the reaction is specific and is a function of both enzyme concentration and time of incubation. It is recalled here that the existence of an enzyme specific for irradiated DNA in M. luteus was first demonstrated by such a biologicad assay (Rarsch, Kamp & Adema, 1964). Recently, it was shown that M. luteus endonuclease is effective in restoring the biological activity of u.v.-inactivated Haemophilus in$uenzae and Bacillus subtilis transforming DNA (Setlow, Setlow & Carrier, 1970; Heijnecker et al., 1971). Wo thank N. Otsuji and WC also thank of T4 excision Promot,ion of
Drs J. Cairns, A. J. Clark, I?. Howard-Flanders, H. Ogawa, Y. Ohshima., K. Shimada for making available to us the bacterial strains usedin this study. Dr S. Okubo for discussion and Mrs S. Ohshima for providing a preparation enzyme. One of the authors (A. T.) is indebted to the Japan Society for th’e Sciences, which enabled him to visit Kyushu University.
14
A. TARETO,
S. YAStJl>-4
ANI>
31. SEKICUCHI
REFERENCES Barbour, S. D., Nagaishi, H., ‘l’cmplin, A. & Clark, .4. ,J. (1970). Proc. Nat. Acad. Sci., Wnsh. 67, 128. Boyce, R. P. & Howard-Flanders, 1’. (1964). Proc. Nut. Acad. Sci., Wash. 51, 293. Boyle, J. M., Paterson, M. C. & Setlow, R. B. (1970). Xature, 226, 708. Boyle, J. M. & Setlow, R. B. (1970). J. ;MoZ. Biol. 51, 131. Carrier, W. L. & Setlow, R. B. (1970). J. Bad. 102, 178. De Lucia, P. t Cairns, J. (1969). Nature, 224, 1164. Friedberg, E. C. & King, J. J. (1971). J. Bact. 106, 500. Grossman, K., Kaplan, J., Kushner, S. & Mahlcr, I. (1968). Cold. Spr. Harb. Symp. Quunt. Biol. 33, 229. Harm, W. (1963). Virology, 19, 66. Heijneker, H. L., Pannekoek, H., Oosterbaan, R. A., Pouwels, P. H., Bron, S., Arwert, F. & Venema, G. (1971). Proc. Nat. Acad. Sci., Wash. 68, 2967. Howard-Flanders, P., Boyce, R. P. & Theriot, L. (1966). Genetics, 53, 1119. Jansz, H. S., Pouwels, P. H. & Van Rotterdam, C. (1963). Biochim. biophys. Acta, 76, 056. Kaplan, J. C., Kushner, S. R. & Grossman, L. (1971). Biochemistry, 10, 3315. Kato, T. & Kondo, S. (1970). J. Bact. 104, 871. Kelly, R. B., Atkinson, M. R., Huberman, J. A. & Kornberg, A. (1969). Nature, 224, 495. Klein, A. & Niebch, U. (1971). Nature New Biol. 229, 82. Kuempel, P. L. & Veomett, G. E. (1970). Biochem. Biophys. Res. Comm. 41, 973. Kushner, S. R., Kaplan, J. C., Ono, H. & Grossman, L. (1971). Biochemistry, 10, 3325. Mahler, I., Kushner, S. R. & Grossman, L. (1971). Nature New BioE. 234, 47. Miura, A. & Tomizawa, J. (1968). Molec. Gen. Genetics, 103, 1. Nakayama, H., Okubo, S. & Takagi, Y. (1971). Biochim. biophys. Acta, 228, 67. Ogawa, H. (1970). MoEec. Gen. Genetics, 108, 378. Ogawa, H., Shimada, K. & Tomizawa, J. (1968). Molec. Gen. Genetics, 101, 227. Ohshima, S. & Sekiguchi, M. (1972). Biochem. Biophys. Res. Comm. 47, 1126. Okazaki, R., Arisawa, M. & Sugino, A. (1971). Proc. Nat. Acad. Sci., Wash. 68, 2954. Okubo, S., Nakayama, H., Sekiguchi, M. & Takagi, Y. (1967). Biochem. Biophys. Res. Comm.27, 224. Okubo, S., Nakayama, H. & Takagi, Y. (1971). Biochim. biophys. Aeta, 228, 83. Rorsch, A., Kamp, C!. V. D. 85 Adema, J-. (1964). Biochim. biophys. Acta, 80, 346. Sekiguchi, M., Yasuda, S., Okubo, S., Nakayama, H., Shimada, K. & Takagi, Y. (1970). J. Mol. Biol. 47, 231. Setlow, R. B. & Carrier, W. L. (1964). Proc. Nat. Acad. Sci., Wash. 51, 226. Setlow, R. B. 8: Carrier, W. L. (1966). Biophys. J. 6, TC2. Setlow, R. B., Setlow, J. K. & Carrier, W. L. (1970). J. Butt. 102, 187. Shimada, K., Ogawa, H. & Tomizawa, J. (1968). Molec. Gen. Genetics. 101, 345. Smith, K. C. & Meun, D. H. (1970). J. Mol. Biol. 51, 459. Strauss, B., Searashi, T. 85 Robbins, M. (1966). Proc. Nut. Acad. Sci., Wash. 56, 932. Takagi, Y., Sekiguchi, M., Okubo, S., Nakayama, H., Shimada, K., Yasuda, S., Nishimoto, T. & Yoshihara, H. (1968). Cold Spr. Harb. Symp. Quunt. Biol. 33, 219. Taketo, A. & Kuno, S. (1969). J. Biochem. 65, 361. Taketo, A. & Kuno, S. (1972). J. Biochem. 71, 497. Yarus, M. & Sinsheimer, R. L. (1964). J. MOE. Biol. 8, 614. Yasuda, S. & Sekiguchi, M. (1970a). J. &!ol. Biol. 47, 243. Yasuda, S. & Sekiguchi, M. (1970b). Proc. Nat. Acad. Sr%., ll~ash. 67, 1839. Willetts, N. S. & Clark, A. J. (1969). J. Bact. 100, 231.
Initial Step of Excision Repair in Escherichiu cd: Replacement of Defective Function of uvr Mutants by T4 Endonuclease V AKIRA
TAKETO?,
SEIICHI
YASUDA
AND MUTSUO
SEKIGUCHI
Department of Biology, Faculty of Science Kyu.shu University, Fukuoka, Japan (Received 24 April
1972)
The ultraviolet sensitivity of double-stranded replicativo form and singlestranded DNA of bacteriophage +A and +X174 was studied by using spheroplasts of various strains of Escherichiu co.%. The replicative form is more sensitive when assayed on uvr- mutants than when plated on wild type or ret- mutants, while t,he single-stranded DNA exhibits the same high sensitivity on these strains. Pretreatment of ultraviolet-irradiated RF$ by a purified preparation of T4 endonuclease V leads to a marked increase in biological activity of DNA when t,he activity is assayed on uvr- mutants but not on the wild type or ret- mutants; the same treatment, however, has no detectable effect on the activity of nonirradiated RF. Thus, the function in vivo controlled by the uvr genes of E. coli can be replaced by the reaction in vitro catalysed by T4 endonuclease V. It is suggested that the first step of excision repair in E. coli is to induce a singlestranded break at a point close to a pyrimidine dimer, possibly to the 5’ side of a dimer, in DNA, and that, the step is controlled by at, least four genes, UWA, uvrB, uvrC and uvrD.
Irradiated RF treated with T4 endonuclease V or pancreatic deoxyribonuclease, or non-irradiated RF treated with the pancreatic enzyme, exhibit lower infectivity when assayed on pal- strains as compared to assaying on pal+ strains. Treatment of irradiated RF by both T4 endonuclease V and excision enzyme, which produces gaps several niicleotides in length in the molecule, also decreased tho I biological act,ivity of RF in poZ- st,rains. It seams that E. co& DNA polymerase is required to repair gaps and nicks in DNA.
1. Introduction Mechanism of repair of DNA by excision has been studied extensively using mutants of Escherichia coli sensitive to ultraviolet radiation. It has been demonstrated that strains having mutation in one of uvrA, uvrB and uvrC genes are unable to excise U.V. induced pyrimidine dimers from DNA; thus, at least three different genes may control the excision process (Setlow & Carrier, 1964; Boyce & Howard-Flanders, 1964; Howard-Flanders, Boyce & Theriot, 1966). It has been shown, moreover, that strains with mutation in uvrD, polA or ret genes degrade their own DNA extensively after U.V. irradiation, suggesting that these genes are responsible for repair synthesis after 1968; De Lucia & Cairns, 1969; Boyle, excision (Ogawa, Shimada & Tomizawa, Paterson & Setlow, 1970; Kato & Kondo, 1970). t On leave from the Department of Biochemistry, Kanazawa University $ Abbreviation used: RF, double-stranded replicative form DNA. 1 1
Medical
School.
2
A. TAKETO,
S. YASUDA
AND
&I. SEKIGUCHI
Although great efforts have been made to elucidate the enzymic mechanism of excision repair in E. coli, no enzyme responsible for excision of pyrimidine dimers has been identified. An endonuclease activity that is specifically active on u.v.-irradiated DNA was found in an extract of E. coli; however, all the mutants so far examined. including those unable to excise dimers in viva, also possessed the enzyme activity (Takagi et al., 1968). We have shown that an extract of E. coli infected with bacteriophage T4 contains a high level of an enzyme activity that induces single-stranded breaks specifically in u.v.-irradiated DNA (Yasuda & Sekiguchi, 1970aJ; Friedberg & King, 1971). The enzyme, T4 endonuclease V, is not, found in an extract of cells infected with u.v.sensitive mutants of T4; thus, it seems probable that the enzyme is responsible for the first step of excision repair in T4-infected cells. A similar endonuclease activity has been found in Micrococcus Euteus (M. lysodeikticus) (Strauss, Searashi & Robbins. 1966; Takagi et aE., 1968; Grossman, Kaplan, Kushner & Mahler, 1968; Carrier & Setlow, 1970; Nakayama, Okubo & Takagi, 1971). The availability of such an endonuclease should make it possible to study the initial steps of excision repair in E. coli. If the first step of excision repair in E. coli is catnlysed by an enzyme similar to T4 endonuclease V, u.v.-irradiated infective viral DNA, once treated in vitro by the T4 enzyme, may be repaired by mutants defective in the incision step (Fig. 1). This paper describes the results of such experiments.
Wild type
-
+
+
+
Mutant I
-
-
+
+
Mutant Ii
-
Mutant IJI
-
-
-
+
-
-
FIG. 1. Schematic representation of the proposed method for the analysis of excision--repo.it mechanism of E. coli. Double-stranded DNA, pre-exposed to u.v., can be repaired by the excisioll+ repair system of E. coli, whereas single-stranded phage DNA cannot. E. coli mutants which at)‘~ defective in one of the steps in repair are unable to repair irradiated DNA. When, howevt,r, irradiated double-stranded DNA is converted to intermediate forms, such intermediates may 1)(x repaired by mutants which are defective in the earlier steps but not in the later ones. ( + ) Repairable; (-) non-repairable.
2. Materials and Methods (a) Bacteria
and bacteriophage
The UVT- mutants used are derived from two distantly related E. coli K12 strain);: N17-9 (u~~A54), N3-1 (uv~B51), N17-7 (uvrC56) and N14-4 (uvrD3) derived from W3623 (Ogawa et al., 1968); and AB1886 (uvrAG), AB1885 (uvrB5) and AB1884 (uurC34) derived
REPAIR
OF DNA
IN
E. COLZ
3
used, AB2463 (recAl3), from AB1157 (Howard-Flanders et aE., 1966). The ret- mutants AB2470 (recBZl), SR88 (recC22), JC5547 (recA13 recB21 recC22), are derivatives of AB1157 (Willetts & Clark, 1969; Smith & Meun, 1970). Double mutants, JC5412 (recB21 $5~8) (Barbour, Nagaishi, Templin & Clark, 1970) and N212 (recA1 uwrA54) (Miura & Tomizawa, 1968), were also used. DNA polymerase I-deficient mutants, P3478 (poEAl) and N611 (pal). are derived from W3110 and W3623, respectively (De Lucia & Cairns, 1969; Ogawa, 1970). Bacteriophage +A is a spherical virus containing single-stranded circular DNA and immunologically related to #X174 (Taketo t Kuno, manuscript in preparation). The phage was propagated in E. coli C and used in most of these experiments. Bacteriophage +X1 74 and T4 were also used in some of the experiments. (b) Enzymes T4 endonuclease V was prepared from T4-infected E. coli 1100 by a modification of the procedure previously described (Yasuda & Sekiguchi, 19705). The purification procedure, details of which will be published elsewhere, includes phase partition in dextran/polyothelene glycol and column chromatography of CM-Sephadex C-25 and of Hypatite C. The enzyme was purified about 1300-fold and is free of other endo- and exonucleases. E. coZi N29 (poZ- endoI- ), was provided by Excision enzyme, isolated from T4iv,-infected Mrs S. Ohshima of this laboratory. The enzyme excises dimer-containing nucleotides from u.v.-irradiated DNA, which has been treated with T4 endonuclease V (Ohshima & Sekiguchi, 1972). Purified bovine pancreatic deoxyribonuclease was obtained from Sigma Chemical CO.
(c) Preparation
of DNA
Infective single-stranded DNA was extracted from purified +A particles by phenol treatment and dialysed against 0.05 M-TriseHCl buffer, pH 8.0 (Taketo I% Kuno, 1969). The +A DNA is converted into double-stranded replicative form upon infection of the host, E. coli C (Taketo & Kuno, 1972). To prepare RF, +A-infected cells were incubated with 25 pg chloramphenicol/ml for 90 min at 37”C, collected and lysed by lysozyme/EDTA/ sodium dodecyl sulphate method. After centrifugation at 100,000 g, the lysate was shaken with 0.05 M-borate-saturated phenol and subjected to isopropanol precipitation. The host DNA was removed by repeated winding with a glass rod and RNA was digested by ribonuclease. After the second phenol treatment and isopropanol precipitation, the material was applied to a methylated albumin column. The RF eluted at 0.65 M-NaCl was collected and dialysed against 0.05 M-Tris.HCl buffer, pH 8.0. RF of 4X174 was also prepared by a similar procedure. Labelled RF was prepared from #A-infected E. coli C thy-, incubated with 2 pg 1hymine/ml. (0.2 &i/pg), as described above.
(d) Irradiation
of DNA
DNA was irradiated with a 15 W Toshiba germicidal most experiments, irradiation was for 25 set at a distance mate dose: 600 ergs/mm2). The dose rate was determined 11.v. inmnsity meter.
lamp at room temperature. In of 25 cm from the lamp (approxiby a Blak-ray J-255 short-wave
(e) Assay of infectivity Infectivity of DNA was assayed on lysozyme/EDTA spheroplasts. Cells were harvested from an exponentially growing culture in 50 ml. of broth, washed with 10 ml’ of 0.01 MTris.HCl (pH 7.5) and suspended in 5 ml. of 20% sucrose/O.1 M-Tris.HCl (pH 8.0) followed by an addition of lysozyme (80 pg/ml.). After 10 min at room temperature, (0.08 ml. of 0.05 M-EDTA (pH 7.5) was added and the mixture was allowed to stand for a further 10 min. The reaction was terminated by addition of 0.1 ml. of 1 M-MgSO, and 0.05 ml. of 1 M-Call, and the spheroplasts were collected by centrifugation at 4000 g foi 10 min in the cold and suspended in 1 ml. of chilled 20% sucrose broth. To determine infectivity of DNA, 0.1 ml. of the spheroplast suspension was added to a mixture of 0.1 ml.
4
A. TAKETO,
S. YARUDA
AND
hr. AEKIQUCHI
of DNA in 0.05 M-‘i’ris.HCl (pH 84) and O-1 ml. of 2UU/, sucrose broth. The mixture was incubated at 37°C for 12 min, diluted with chilled 20% sucrose broth and then plated, at. least in duplicate, with a culture of E. co& C in top agar, which consists of 20% sucrose:/ 0.5% agar/broth. Throughout the process care was taken to avoid photoreactivation. (f) Determination
of pyrimidine
dimers
DNA and the acid-soluble fractions were hydrolysed by heating acid at 100°C for 3 hr. The amount of thymine and thymine-containing mined by Dowex-1 column chromat,ography (Sekiguchi et al., 1970).
with 6 N-perchloric dimers was deter-
3. Results (a) Host cell reactivation
of irradiated
replicutive
fornz DNA
Figure 2 indicates the u.v.-sensitivity of single- and double-stranded DNA of $A assayed on spheroplasts of various E. coli strains. The RF is more sensitive when assayed on u.v.-sensitive mutants having mutation in one of the uvr genes than on the wild-type strain (Fig. 2(a)). This is in agreement with the results obtained with $X174 RF (Jansz, Pouwels & Van Rotterdam, 1963; Yarus & Sinsheimer, 1964). As shown in Figure 2(b), there is a small but consistent difference between u.v.-sensitivity of the RF assayed on pal + and on pal- strains as observed with $X174 (Klein & Niebch, 1971); irradiated RF exhibits lower survival in DNA polymerase-deficient mutants, P3478 and N611, isolated from the different parents. On the other hand, no difference in u.v.-sensitivity is observed when the RF is assayed either on ret + or on ret - strains (Fig. 2(c)), though the ret- mutants themselves are sensitive to WV. In spite of the great difference of u.v.-sensitivity of the RF, single-stranded DNA exhibits almost an equally high u.v.-sensitivity when assayed on spheroplasts of various strains. These results are consistent with the idea that the RF of $A can be reactivated by a host cell mechanism, in which the double-stranded nature of DNA is required for repair of u.v.-induced lesions by excision and resynthesis. It appears that, E. coli genes uvrA, uvrB, uvrC and uvrD as well as polA are involved in the process. whereas recA, recB and recC are not.
(b) Effect of T4 endonuclease
V on the reactivation
of replicative
form DNA
If excision repair proceeds in sequential manner and the host cell reactivationnegative mutants are defective in one of the steps, intermediates in the repair process should be reactivated by mutants which are defective in the earlier steps but not in the later ones (see Fig. 1). In order to test this possibility, experiments were performed using T4 endonuclease V as an enzyme to produce such an intermediate. It has been demonstrated that the enzyme induces a single-stranded break at a point close to a pyrimidine dimer (Yasuda & Sekiguchi, 1970b). In an experiment, the result of which is shown in Figure 3, +A RF was irradiated with various doses of U.V. and then incubated with or without T4 endonuclease V at, 37°C for 30 min in 0.04 M-Tris*HCl, pH 7.5. After appropriate dilution, the DNA’s were mixed with spheroplasts of wild-type E. coli W3623 and of its uvrA- mutant, N17-9, and infectivity was determined. As can be seen in the Figure, preincubation of irradiated RF with T4 endonuclease V leads to a marked increase of biological activity of the RF when the activity is assayed on the uvrA- mutant but not on the wild-type strain; the same treatment, however, has no detectable effect on the activity
’ +. \1’ \ \ A ‘1b\
(0)
lh
i
(b)
1..
10-I - “a\ \ A\A ‘1\ g \ \\ \0 8 A\ :\ lo+t ‘1\ \\ \ “\ \o .\ \\ 1O-3: \\ \ .-6 ‘?\ B I I I I -F lo-4 ‘5 .-> c (cl ‘;;\. \\ -----I--b \\ ‘4.
\
\
i‘, ‘? \
‘? /AL 5
IO
u v. Irradmtlon
15
20
time (set)
\* \
1
I
I
I
5
IO
15
20
uv. Irrodiatton tipe (set)
2. Host cell reactivation of $A RF. RF (3.8 x lo4 plaque-forming units/ml. on E. coli C spheroplast) or single-stranded DNA (3.8 x lo6 plaque-forming units/ml. on E. coli C spheroplast) of +A was irradiated at a distance of 25 om from a 15 W germicidal lamp (Toshiba) and the infectivity was assayed on spheroplasts of various E. coli strains. (a) uvr System: (0) W3623 (wild); (0) Nli’-9 (uwrA); (A) N3-1 (uvrB); ( q i) N17-7 (uurC); (A) N14-4 (uvrD). (b) ~oZ system: (0) W3623 (wild); (A) N611 (pal); (A) P3478 (poZA). (c) ret system: (0) W3623 (wild); (011 AB2463 (recA); (a) AB2470 (recB); (0) SR88 (recC). (-) RF; (----) single-stranded DNA.. FIG.
G
A. TAKETO,
S. YASUDA
,0-l 0
,:
AND
I
IO 20 u v. Irrodation
M. SEKIGUCHI
I
1
30 tune (SK)
40
on the infectivity of u.v.-irradiated RF. RF or FIG. 3. Effect of T4 endonucleaae V treatment single-stranded DNA of $A was irradiated, as described in Fig. 2, and then incubated with or without T4 endonuclease V at 37°C for 30 min in 0.04 M-Tris .HCl buffer, pH 7.5. After suitable dilution, the infectivity was assayed on W3623 (wild) or N17-9 (uurA) spheroplasts. ( 0) Untreated DNA, assayed on W3623; (A) untreated DNA, assayed on N17-9; (0) enzyme-treated DNA, assayed on W3623; (A) enzyme-treated DNA, assayed on N17-9. ((--) RF; (----) singlestranded DNA.
of non-irradiated RF. This indicates that the reaction in vitro catalysed by T4 endonuclease V replaces, to a certain extent, the function in viva controlled by the uvrA gene of E. co&. (c) Comparison of reactivation
capacities
of E. coli mutants
The abilities of various E. coli mutants to reactivate irradiated RF which has been treated with T4 endonuclease V were next compared. The results, summarized in Tables 1 and 2, indicate that uvrA-, uvrB- and uvrC- mutants respond strongly to the enzyme treatment; 15 to 75fold increase of the biological activity is observed with both the AB and N series of uvr- mutants, isolated from AB1157 and from W3623, respectively. A small but significant reactivation was observed with uvrDmutant. As expected, ret- mutants, which have normal capacity for reactivation, do not respond to the enzyme treatment. Of interest is the observation that pal- mutants exhibit lower survival when irradiated RF has been treated with the enzyme. Effect of T4 endonuclease V is specific for irradiated RF. When non-irradiated RF is incubated with the enzyme, no significant change in the biological activity is observed, irrespective of the types of mutants employed (Table 1). Moreover, it was shown that treatment of irradiated RF with pancreatic deoxyribonuclease has little effect on the activity assayed on various hosts (Table 2). The pancreatic enzyme is known to induce single-stranded breaks, having no specificity for pyrimidine dimers. This will be discussed later in more detail. The kinetics of the reaction are shown in Figure 4. In these experiments the RF was irradiated at the fixed dose (for 25 seconds at a distance of 25 cm from a lamp;
REPAIR
OF DNA
IN
7
E. COLI
TABLE 1 Effect of T4 endonucleu.se V treatment on infectivity of u.v.-irradiated or non&m&ted +A RF Relative infectivity after T4 endonuclease V treatmentt
Surviving fraction of irradiated RF
Strains
AB1157 (wild) AB1886 (uvrA) AB1885 (uurB) AB1884 (uwrC) JC5547 (recA recB recC) JC5412 (recB &A) N212 (recA uurA) P3478 (poZA)
Irradiated RF
2.2 x 10-l 2.1 x10-s 2.4~10-~ 3.8 x 1O-3 2.3 x10-i 2.1 x10-i 7.8~10-~ 8.7 x 1O-2
1.00 15.7 75.0 29.7 1.06 I.02 36.7 0.41
Non-irradiated RF 0.93 0.96 0.99 I.05 1.10 1.00 1.16 1.00
,$A RF was irradiated et a disttmce of 25 cm from the lamp for 25 set and divided into two portions. One portion was incubated with T4 endonucle~e V (0.4 pg protein/ml.) in 0.05 M-Tris. HCl (pH 7.5) et 37°C for 10 mm, 8nd the other was incubated without the enzyme. Non-irradiated RF was also divided into two portions and treated in the same manner. The four types of sample were subjected to infectivity assay using spheroplasts of various E. coli strains. + Ratio
of T4 endonuclease
V-treated
RF to untreated
RF.
TABLET Effect of treatment with T4 endonuclease V or pancreatic deoxyribonuclease on infectivity of irradiated $A RF
Strains
W3623 (wild) N17-9 (uurA) N3-1 (uvrB) N17-7 (WC) NlP4 (uurD)
Surviving fraction of irradiated RF 1.0x 2.0x 2.3 x 1.3 x 1.7 x
10-i 10-3 1O-3 10-s IO-2
Relative
infectivity of irradiated RFt after treatment with Pancreatic T4 endonuclease V DNase 0.90 34.6 43.7 40.1 3.63
0.89 0.67 1.00 0.75 0.80
4A RF was irradiated with U.V. at B distrmce of 25 cm for 25 set and divided into three portions: (1) treated with T4 endonuclease V (0.4 pg protein/ml.) in 0.05 M-Tris.HCl (pH 7.5) at 37’C for 10 min; (2) treated with pancreatic DNase (0.001 pg/ml.) in 0.05 M-Tris .HCl/O.Ol nr-MgSo, (pH 7.5‘) at 37°C for 10 min; (3) without treatment. The three types of samples were subjected to infectivity assay using spheroplasts of various E. coli strains. t Ratio of enzyme-treated RF to untreated RF.
approximately 600 ergs/mm2) and treated with T4 endonuclease V at different concentrations for 10 minutes (Fig. 4(a) ) or at a concentration of O-4 pg protein/ml. for different times (Fig. 4(b)). The reaction was terminated by shaking with phenol and the DNA, recovered from the aqueous layer and dialysed, was assayed on spheroplasts of various strains. The mutants examined were uvrA-, uvrB- and uvrC-> derived from AB1157, and P3478 (~oZA) (Fig. 4(a)), and uvrA-, uvrB-, uvrC-, uvrD--
8
A.
TAKETO,
1 01
1o-3 ’ 0
I. 02
Concentration
S. YASUDA
I-/: 03
04
AND
05
0
of enzyme (~9 protein/ml
)
M.
SEKIGUCHI
5
I IO
I 15
I 20
Ttme of mcubatlon(min)
FIG. 4. Kinetics of the reaction. (a) Effect of concentration of T4 endonuclease V. RF, irradiatecl was incubated with various amounts of T4 endonuclease V at 37°C for 10 min at 600 ergs/md, in 0.05 iv-Tris.HCl, pH 7.5. After shaking with phenol, the DNA was dialysed and assayed on spheroplasts of the following strains. (0) AB1157 (wild); (0) AB1886 (uwTA); (A) AB188R (uvrB); (0) AB1884 (u&Z); ( n ) P3478 (poZA). (b) Time-course of the reaction. RF, irradiated at 600 ergs/mm2, was incubated with 0.4 pg protein/ml. of T4 endonuclease V at 37°C in 0.04 x-T&. HCl, pH 7.5. At the times indicated, the reaction was terminated by shaking with phenol and thP biological activity was assayed, after dialysis, on spheroplasts. (0) W3623 (wild); (0) N17-9 (uvrA); (A) N3-1 (uvrB); (0) N17-7 (uvrC); (A) N14-4 (uvrD); ( n ) N611 (pal).
and pal-, derived from W3623 (Fig. 4(b)). F rom the results, it is evident that t’he reactions are functions of both time and enzyme concentration. With increasing time of incubation or concentration of enzyme the activities assayed on uvr- mutants increased, while those assayed on pal- mutants decreased. At the plateau, some uvrmutants exhibit almost the same high level of reactivation as do the wild-type strains. It is worthy of note that uvrD- mutant is able to reactivate the enzyme-treated RF. t,hough the mutant differs from other uvr- mutants in many properties (Ogawa et al., I 968). TABLE
Effect of T4 endonuclease
1’ treatment or non-irradiated
W3623 (wild) Nl7-9 (uvrA) N3-1 (uvrB) N17-7 (WC) N14-4 (uvrD)
on infectivity $X174 RF
of u.v.-irradiated
Relative infectivity after T4 endonuclease V treatmenti
Surviving fraction of irradiated RF
Strains
3
Irradiated RF
2.0 x 10-l 3.0 x 10-S 3.4x10-3 3.2x 1O-3 2.8 x 1O-2
1.8 24.3 54.5 17.5 4.5
Non-irradiated RF 0.93 0.78 0.83 0.87 I.08
The conditions and procedures were the same as described in the legend to Table 1 except +X174 RF was treated with a higher concentration (12 pg protein/ml.) of T4 endonuclease t Ratio
of enzyme-treated
RF
to unt,reated
RF,
that V.
REPAIR
OF DNA
IN
E. COLI
Similar results were obtained with RF of $X174; a considerable place when irradiated +X174 RF were treated with T4 endonuclease one of the four types of uvr- mutants (Table 3). (d) Infectivity
9 reactivation took V and assayed on
of RF with nicks or gaps
The foregoing experiments demonstrated that pre-treatment of irradiated RF b;y T4 endonuclease V results in a decrease of biological activity of DNA when the activit,y was assayed on pal- mutants. Since no such decrease was observed when the assa,y was made on the wild-type strain, it was suspected that nicks produced in RF by the endonuclease are more injurious in pal- cells than in pal+ cells. In order to test this possibility, we have investigated the effect of pancreatic deoxyribonuclease on the infectivity of RF in W3623 (poZ+) and N611 (pal-). As shown in Figure 5, the rate of I
Time of mcubation (min)
FIG. 5. Inactivation of u.v.-irradiated and non-irradiated RF by pancreatic deoxyribonuclease. +A RF, irradiated with u.v. (at 600 ergs/mm2), or non-irradiated RF, was incubated at 37°C wii,h 0.005 pg/ml. of pancreatic deoxyribonuclease in 50 mM-Tris*HCl/lO mM-MgSO,, pH 7.5. Samples were withdrawn at the times indicated and infectivity was assayed on spheroplasts of W3623 and RF on N611; ( 0) irradiated RF on N611. (0) Non-irradiated RF on W3623; (A) non-irradiated W3623; (A) irradiated RF on N611.
inactivation of both irradiated and non-irradiated RF is higher in the pal- strain than in the pal+ strain. It was shown, moreover, that irradiated RF is inactivated by thr enzyme more rapidly than is non-irradiated RF, probably due to introduction of additional nicks into irradiated RF by certain endonucleases specific for irradiated DNA in E. coli. These results are consistent with the idea that DNA polymerase I is involved in repair of nicks produced in RF by the enzyme treatments. Recently, it was shown that the combined action of T4 endonuclease V and excision enzyme results in the excision of dimer-containing nucleotides from u.v.irradiated T4 DNA (Ohshima & Sekiguchi, 1972). The result shown in Table 4 indicates that irradiated +A RF is also subjected to the action of these enzymes. On incubation with T4 endonuclease V or excision enzyme alone, no appreciable
45
0 1 1
(cts/min)
Acid-soluble
38
0 0.8 0.7
(Oh release)
Dimers
39
0 15 3
(cts/min)
fraction
2.5
0 0.9 0.2
(% release)
Thymine
73
112 126 142
(cts/min)
Dimers
0.75 0.77 0.77 0.47
15,350
dimers (%)
Content
14,900 16,150 17,900
(ctsjmin)
DNA fraction Thymine
of
[‘*C]Thymine-labelled 4A RF was irradiated at 600 ergs/mm2 and incubated at 37°C for 60 min. The reaction mixture (0.3 ml.) contained 0.2 pg of DNA, 12 pmoles of Tris-maleate (pH 7.0), 3 pmoles of MgCl, and T4 endonuolease V (0.2 pg) and/or excision enzyme (1.05 pg). After incubation, the mixture was acidified and separated into DNA and acid-soluble fraction. Each sample was hydrolysed by heating with 6 rr-perchloric acid at 100°C for 3 hr and the radioactivity of thymine and its dimers was measured after separation by Dowex 1 column chromatography.
None Endonuclease V Excision enzyme Endonuclease V plus excision enzymes
Enzyme
4
Effect of T4 endonuclease V and excision enzyme on the release of pyrimidine dimers from u.v.-irradiated +A RF
TABLE
REPAIR
OF DNA
IN
E. COLI
11
amount of dimers or thymine is released from the DNA. When, however, both enzymes are present in the reaction mixture, selective release of dimers takes place; about 40% of the dimers originally present in the DNA become acid-soluble, while only 3% of the thymine is released from the DNA. From the ratio of radioactivity in thymine and its dimers in the acid-soluble fraction and the thymine content of DNA, it is estimated that about six nucleotides are excised for each dimer. Thus, the RF treated by the two enzymes would possess gaps of several nucleotides in length in the molecule. Infectivity of RF treated with these enzymes was next investigated. As shown in Table 5, irradiated replicative forms, treated with T4 endonuclease V alone or with TABLE
5
Effect of T4 endonucleme V and excision enzyme on the infectivity of u.v.-irradiated q5A RP Infectivity W3623
Enzyme
None Endonucleese V Excision enzyme Endonuclease V plus excision enzyme QA RF w&s irradiated at 600 (0.3 ml.) contained irradiated endonucleaae V (3.7 pg) and/or W3623 or N611, w&s expressed
assctyed on N611
22.7 49-o 33.2
14.4 2.1 17.1
49.4
0.9
ergs/mn? snd incubated at 37’C for 10 min. The incubation mixture RF, 15 pmoles of Tris-HCl (pH 7.5), 3 pmoles of MgSO, and T4 excision enzyme (1.72 pg). Infectivity, sswyed on spheroplasts of 88 y. of non-irradiated RF (without enzyme treatment).
both endonuclease V and excision enzyme, exhibit almost the same low infectivity for pal- mutant. No such decrease of infectivity was observed when the treated RF was assayed on poZ+ strain. Thus, gaps as well as nicks in the DNA appear to be equally injurious in pal- cells.
4. Discussion Endonucleases which act specifically on DNA pre-exposed to U.V. radiation have been found in several micro-organisms. First, the activities were detected in extracts of M. lutew and also of E. coli (Strauss et al., 1966; Grossman et al., 1968; Takagi et al., 1968). The enzyme was purified from M. luteus and its properties have been studied extensively (Carrier & Setlow, 1970; Kaplan, Kushner & Grossman, 1971; Nakayama, Okubo & Takagi, 1971). Although the endonucleases have many characteristics expected of an enzyme that catalyses the first step of excision repair, the role of the enzymes in in vivo repair has not fully been understood. A number of u.v.-sensitive strains from E. coli and from M. 1uteu.s possess the normal level of endonuclease activities, while mutants lacking the enzyme are only slightly u.v.-sensitive (Okubo, Nakayama, Sekiguchi & Takagi, 1967; Takagi et al., 1968; Okubo, Nakayama cc5 Takagi, 1971; Mahler, Kushner t Grossman, 1971).
12
A. TLKETO,
S. YASUDA
ANI)
11. SEKIGT:(‘HT
Recently, it was found that T4 endonuclease I#*, which is similar to X. luteu.~ endonuclease in many respects, is induced in E. coli after infection with T4 (Yasuda $ Sekiguchi, 1970a$; Friedberg & King, 1971). The formation of the enzyme is controlled by the v+ gene of T4, the gene which is known to increase survival of phage after U.V. irradiation (Harm, 1963). It was shown, moreover, that excision of pyrimidine dimers in viva as well as in vitro takes place in T4v+ -infected cells but not in T4v--infected cells (Setlow & Carrier, 1966; Sekiguchi et al., 1970; Katsuki &Sekiguchi, manuscript in preparation), Thus, it seems probable that the enzyme is responsible for the first step of excision repair in T4-infected cells. In the present investigation, we have demonstrated that pre-treatment of u.v.irradiated double-stranded viral DNA by a purified preparation of T4 endonuclease \ leads to a marked increase of biological activity of DNA when the infectivity was assayed on E. eoli strains having mutation in the uvr,4, B, C or D gene. Since these mutants are unable to reactivate irradiated RF without the enzyme treatment, it ia suggested that T4 endonuclease V supplies a function that is missing in tbtb uvr mutants. It seems probable, therefore, that the first step of excision repair in E. coli is catalysed by an enzyme whose mode of action is similar to that of T4 undonuclease V. There is some evidence that uvrA, B and C genes control some step of the excision process. It has been demonstrated that strains with mutation in one of these genes are unable to excise pyrimidine dimers (Setlow & Carrier, 1964; Boyce 8~ HowardFlanders, 1964; Howard-Flanders et aE., 1966). It has been shown, moreover, that fragmentation of cellular DNA after u.v.-irradiation is less in these mutants than in the wild-type strain (Shimada, Ogawa & Tomizawa, 1968). The present finding that irradiated RF, once treated with T4 endonuclease V in vitro, can be reactivated b> uvr- mutants is in accord with these observations. We had not, however, expected reactivation of the enzyme-treated RF also to take place in the uvrD- mutant. Since the mutant exhibits an extensive degradation of DNA after u-v.-irradiation, it n-as suspected that the mutant is defective in the second or the later steps of excision repair (Shimada et al., 1968). This implies that the uvrD gene function is different. from the functions of the uvrA, B and C genes, even though all these genes may control the incision step. The involvement of at least four genes in controlling the incision step, together with the fact that there is no direct correlation between the u.v.-sensitivity and the in vitro enzyme activity in E. coli, strongly suggests that the process of excision repair in normal E. coli is more complex than that found in T4-infected cells. It is supposed that the repair in infected cells is carried out in the cytoplasm, where the efficiency of the reaction mostly depends on the amount and activity of enzymes involved. If we assume that the repair in normal cells takes place in some particular part of the cell, e.g. cell membrane, it is easily understood that other factors affecting localization and transportation of enzyme and substrate are much more important (Boyle & Setlow, 1970). It has been demonstrated that T4 endonuclease V introduces a break at the 5’ side of a pyrimidine dimer in irradiated DNA (Yasuda & Sekiguchi, 1970b). Thus, if t,he first step of dimer excision in E. coli is catalysed by an enzyme similar to T4 endonuclease V, then the enzyme that is responsible for the second step should be an exonuclease that hydrolyses a polynucleotide in a 5’ + 3’ direction, or else a specific endonuclease that induces a break to the 3’ side of a dimer (Fig. 6). It was demon-
3’
REPAIR
OF DNA
Endonucleose
V
IZ. COLI
5’
P
z
i 5’ 3’ FIG.
IN
6. A possible scheme for the initial
a Exonucleose b-3’) or Endonucleose
I4
5’
3’
steps in the repair of u.v.-irradiated
DNA.
strated that DNA polymerase I of E. coli possessesa 5’ + 3’ exonuclease activity and can excise dimer-containing nucleotides from irradiated DNA in vitro (Kelly, Atkinson, Huberman & Kornberg, 1969). It might be possible that both the second step of excision and repair replication are catalysed by a single enzyme, DNA polymerase I. However, no direct evidence has been obtained to indicate that DNA polymerase 1 is involved in excision of dimers in vivo. The rate and final extent of dimer excision are almost the same in the pal+ and POE- strains (Boyle et al., 1970; Katsuki & Sekiguchi, manuscript in preparation). Thus, there is a possibility that other enzyme(s) function t,o excise dimers or replace DNA polymerase I in the process. The present finding that the u.v.-sensitivity of #A RF inpol- mutants is low compared to that in uvr- mutants also supports this view. Recently, enzymes that catalyse the selective release of dimers from irradiated DNA, pre-treated with endonucleases, were isolated from M. luteus and T4-infected E. coli (Kushner, Kaplan, Ono & Grossman, 1971; Ohshima & Sekiguchi, 1972). It is evident that, apart from functioning in the excision of dimers, DNA polymerase I plays some role in repair of DNA. It has been shown in the present paper that RF possessing nicks or gaps exhibits lower survival in pal- mutants than in the wildtype strain. This implies that DNA polymerase I may be required for filling gaps and sealing nicks in DNA. Recently, it was demonstrated that joining of newly replicated DNA chains in viva is slow in pal- mutants (Kuempel & Veomett, 1970; Okazaki, Arisawa & Sugino, 1971). Finally, it is pointed out that the procedure employed in the present investigation is potentially useful for a specific assay of endonuclease that acts on irradiated DNA. It was demonstrated that the reaction is specific and is a function of both enzyme concentration and time of incubation. It is recalled here that the existence of an enzyme specific for irradiated DNA in M. luteus was first demonstrated by such a biologicad assay (Rarsch, Kamp & Adema, 1964). Recently, it was shown that M. luteus endonuclease is effective in restoring the biological activity of u.v.-inactivated Haemophilus in$uenzae and Bacillus subtilis transforming DNA (Setlow, Setlow & Carrier, 1970; Heijnecker et al., 1971). Wo thank N. Otsuji and WC also thank of T4 excision Promot,ion of
Drs J. Cairns, A. J. Clark, I?. Howard-Flanders, H. Ogawa, Y. Ohshima., K. Shimada for making available to us the bacterial strains usedin this study. Dr S. Okubo for discussion and Mrs S. Ohshima for providing a preparation enzyme. One of the authors (A. T.) is indebted to the Japan Society for th’e Sciences, which enabled him to visit Kyushu University.
14
A. TARETO,
S. YAStJl>-4
ANI>
31. SEKICUCHI
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