J. Mol. Biol. (1976) 102, 15-26
Studies on Temperature-dependent Ultraviolet Light-sensitive Mutants of Bacteriophage T4 : The Structural Gene for T4 Endonuclease V KENZO
SATO
Department
AND
MUTWO SEKIGIWHI
of Biology, Faculty Kyushu University Fukuoka 812, Japm
(Received 12 Jwne 1975, ad
,in revised
fom
qf Scimce
24 November 1975)
Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42°C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30°C they exhibit surviva.1 curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the 2)gene. Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 3O”C, but no selective release of dimers was induced at 42°C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infect,ed cells when the purified enzymes were incubated in a buffer at 42°C. From these results it is evident that the 21gene is the structural gene for T4 endonucleaso V, which plays an essential role in the excision-repair of ultraviolet. light-damaged DNA. The time of action of the repair endonucleasa was determined by using the mutant. Survival of a temperature-sensitive u mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30% for at least ten minutes and then transferred to 42°C. It appears that repair of DNA proceeds during an early stage of phage development.
1. Introduction Genetic studies with radiation-sensitive mutants of bacteriophage T4 revealed that there are at least two repair processes for dealing with ultraviolet light-induced damage in T4 DNA (Harm, 1963; Boyle C%Symonds, 1969). The first process is excision-repair, in which a damaged portion of DNA is excised and repaired by gapfilling and rejoining reactions. It has been shown that T4v mutants are unable to induce T4 endonuclease V, which plays an essential role in the excision of pyrimidine dimers (Yasuda & Sekiguchi, 1970a; Friedberg & King, 1971). The second process bears some resemblance to the type of post-replication repair or recombination repair, although the precise mechanism is as yet unknown (Rupp & Howard-Flanders, 1968; Symonds et al., 1973). T4x and y mutants appear to be defective in this process. In spite of much knowledge on the u gene fun&ion, it is unclear whether the z gene determines the structure of endonuclease V, or controls bhe formation of the 16
16
K.
SAT0
ANL)
111. SEKIGUL‘HI
enzyme in some indirect manner. To understand t,he repair mecha,nism more precisely, we tried to isolate a new type of ultraviolet, light-sensit,ive mutant. In t,hese experiments we were able to isolate mutants with temperature-sensitive mutations in the v gene. Since the mutants produce thermolabile endonuclease V, the u gene is the structural gene for T4 endonuclease V. A preliminary account of this paper has been reported (Sato & Sekiguchi, 1972).
2. Materials and Methods (a) Bacteria
and bacteriophage
coli strain B was used as the phage host. E. coli strain 1100, an endonuclease I defective mutant of K12 (Diirwald & Hoffman-Berling, 1968), was used for experiments Escher&h&z
in vitro. Bacteriophage 1963), were obtained Drake.
T4D and its ultraviolet from Dr W. Harm. T4p2
light-sensitive mutants, v1 and z (Harm, (Drake, 1973) was provided by Dr J. W.
(b) Media (i) M9 medium: NasHP0,.12H,O, 14.7 g; KH2P0,, 3 g; NaCl, 5 g; 0.1 M-MgS04, 10 ml; 0.01 M-CaCl,, 10 ml; 40% glucose, 10 ml in 1 1 of distilled water. (ii) Dilution fluid: M9 medium without glucose, CaCl, and MgSO,. (iii) Broth: polypeptone, 10 g; extract of fish meat, 5 g; NaCl, 3 g in 1 1 of distilled water. (c) Irradiation
and plating
of phages
Phages were suspended in dilution fluid and irradiated with a 15 W Toshiba germicidal lamp at room temperature. In most experiments, irradiation was at a distance of 80 cm of irradiated phage was from the lamp (approx. dose rate 0.6 J/ m2 per s). The plating performed in a dimly lit room to avoid photoreactivation. For control of temperature, plates were pm-incubated at desired temperatures and, after plating, placed quickly into an air-circulating incubator. In some experiments, plates were wrapped in a vinyl sheet and placed in a temperature-controlled water-bath incubator. (d) Isolation
of mutants
T4D wild-type
was mutagenized with 0.4 M-hydroxylamine in 0.05 M-sodium phosphate (Tessman, 1968). After incubation at 37°C for 48 h (survival of 10V2), the suspension was diluted and portions were added to a l-ml culture of E. coli B (1 x lo7 cells/ml) in broth and were incubated at 37°C overnight. In this step, mutants which usually occur in mixed clones segregate from the wild type. The lysates thus obtained were screened for u.v.-sensitive mutants by comparing survival after U.V. irradiation. To avoid selecting mutants originating from t,he same clone, a number of lysates were prepared and only one mutant was isolated from each lysate. The lysates were diluted and plated with E. coli B. Plaques wore picked up with capillaries which were then placed in 1 ml of dilution fluid. Three O.l-ml portions of the suspension were added to holes (6 mm diam.) in a plastic tray, and irradiated with U.V. light at various doses (0, 10 and 20 s at 80 cm from the lamp). By means of a sterile paper strip (5 mm x samples were applied to a plate, which had 70 mm) dipped into the phage suspensions, been seeded with about 10s cells of E. coli B in 3 ml of soft agar and pm-incubated at 42°C for 1 h. After administering phages tho plate was quickly placed in an air-circulating incubator at 42°C overnight. Approximate survival rates of phages were determined by the spot tests, and strains which exhibit increased U.V. sensitivity were selected and further tested for their U.V. sensitivities at 30°C and 42°C by an ordinary plaque-counting method.
buffer (pH 6*0), 0.001 M-EDTA
(e) Assay
of enzyme activities
Excision of pyrimidine dimers in vitro was determined as described by Sekiguchi et al. (1970). Sedimentation analysis of DNA in alkaline sucrose was performed as described previously (Yasuda & Sekiguchi, 19705).
~LTHAirlOLE’J’
LIGHT-YEXS11‘IYE: (f ) Preparatioll.
M[!1’A,L”1’S
OF ‘P-1
1;
of enzyme
E;. co/i 1100 was grown in broth at 30°C’ to a concentration of I x 10s cells/ml iufectrd with strains of T4 at, a mult’iplicity of infect’icln of 4. At 20 min after infection,
a~ltl the culture was chilled rapidly by pouring ont’o crushed ice. (-‘ells were collected by refrigerated cmt,rifugation, resuspended in a buffer containing 0.05 >r-Tris.HC’l (pH 7.5), 0.001 %I-?IYrrt)rokt~r1 cells and dnbris mercaptoethanol and disrupted in an ultrasonic disilltegrator. fraction was \vere rcmovod by crntrifugat,iotr at 10.000 g fol- 20 mill. antI ttle sli])ernatant Ilsed as a cell-frte rxtract.
A 1 -ml sample of bacterial culture exposed to 32P \vas \vit,hdraM.u and acidified iltmlc[l2 ml iately with trichloroacetic acid (final concn. 5”;). Thcr~ 0.1 mmol sodium phosphate. of an overnight culture of E. coli B and 2 x IO’O particlvs of T4D were added to the rnixturct as carriers. After extraction of acid-soluble materials, pllospbolipid and RNA, DNA was I !W). Tllr~ radioactivit>, of DNA uxs ext’ractod by hot trichloroacetjic acid (Sekigllcahi. determined in a gas-flow count,er.
3. Results (a) Isolution
of tenbpernt,ule-de~et~aet~,t
ultrar~iolet
light-.wtt,.sitice
mutanf.u
(1963) and Boyle t Symonds (1969) isolated u.v.-sensibivr mutants of 1’4 Harm with mutations in three different genes, V. z and y. We have tried t’o isolat’e a Ned type of u.v.-sensitive mutant by selecting mutants at high temperature. To ensur(a obtaining high frequency of mutation, T4 was mutagenized with hydrox.vlamine and u.v.-sensitive mutants were selected. Among 830 plaques examined, ten mutants were isolated ; five of them were temp~raturt,-dt,penderlt, u.r.-sensitive mutants. and the other five were ordinary u.v.-sensitive mutants. whose sensitivity was not significantly affected by temperature. Figure 1 shows survival curves of the tcmperature-dependent mutants and of the reference strains, T4D. and ‘vJ~.-it 42°C the mutants (F81. F431. F794. I?801 and F821) ~‘ere as sensitive as vI, but at 3O’C these mutants displayed a U.V. sensitivity identical to that of thr wild-type. The u.v. sensitivities of T4D and v1 are not significantly affected by t,emperature. When irradiated phages are plated at various temperat,uren. the mut,ants exhibit increased U.V. sensitiviby with elevating t’emperaturr and t hrh sharpest increase iI1 the sensitivity t,akes place between 34°C and 37,-C.
Among u.v.-sensitive T4 mutantSs. r and !/ arc’ sc&tivr to methyl methanesulfonate. whereas v is as resistant t’o t#he agent as t’he wild-type (Mortelmans & Friedberg. 1972; Ebisuzaki et ul., 1975). Thus. the mutants isolated were examined for their methyl methanesulfonate sensitivity. Phages were suspended at 1 x 1O8 plaque-forming units/ml in M9 medium containing 0.01 M-methyl methanesulfonatc and incubatted at’ 37°C. The susptbnsion was diluted at various times (20 to 60 min)
IS
Ii.
S:\‘I’O
.lNL)
\
I IO
\
I 20 Ultravlalet
(a)
Al. SEKIGU(‘HI
I IO
0 irradiation
I 20
time (s) (b)
FIG. 1. Ultraviolet light irradiation survtval CUWHS of T4 mutants. Phages (6 were irradiated at 80 cm from a lamp and plated with E. coli H on pro-warmed werr incubated at, 30°C (a) or 42°C (b). The same result was obtained when E. ashost, (-~-‘:-
Studies on Temperature-dependent Ultraviolet Light-sensitive Mutants of Bacteriophage T4 : The Structural Gene for T4 Endonuclease V KENZO
SATO
Department
AND
MUTWO SEKIGIWHI
of Biology, Faculty Kyushu University Fukuoka 812, Japm
(Received 12 Jwne 1975, ad
,in revised
fom
qf Scimce
24 November 1975)
Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42°C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30°C they exhibit surviva.1 curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the 2)gene. Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 3O”C, but no selective release of dimers was induced at 42°C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infect,ed cells when the purified enzymes were incubated in a buffer at 42°C. From these results it is evident that the 21gene is the structural gene for T4 endonucleaso V, which plays an essential role in the excision-repair of ultraviolet. light-damaged DNA. The time of action of the repair endonucleasa was determined by using the mutant. Survival of a temperature-sensitive u mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30% for at least ten minutes and then transferred to 42°C. It appears that repair of DNA proceeds during an early stage of phage development.
1. Introduction Genetic studies with radiation-sensitive mutants of bacteriophage T4 revealed that there are at least two repair processes for dealing with ultraviolet light-induced damage in T4 DNA (Harm, 1963; Boyle C%Symonds, 1969). The first process is excision-repair, in which a damaged portion of DNA is excised and repaired by gapfilling and rejoining reactions. It has been shown that T4v mutants are unable to induce T4 endonuclease V, which plays an essential role in the excision of pyrimidine dimers (Yasuda & Sekiguchi, 1970a; Friedberg & King, 1971). The second process bears some resemblance to the type of post-replication repair or recombination repair, although the precise mechanism is as yet unknown (Rupp & Howard-Flanders, 1968; Symonds et al., 1973). T4x and y mutants appear to be defective in this process. In spite of much knowledge on the u gene fun&ion, it is unclear whether the z gene determines the structure of endonuclease V, or controls bhe formation of the 16
16
K.
SAT0
ANL)
111. SEKIGUL‘HI
enzyme in some indirect manner. To understand t,he repair mecha,nism more precisely, we tried to isolate a new type of ultraviolet, light-sensit,ive mutant. In t,hese experiments we were able to isolate mutants with temperature-sensitive mutations in the v gene. Since the mutants produce thermolabile endonuclease V, the u gene is the structural gene for T4 endonuclease V. A preliminary account of this paper has been reported (Sato & Sekiguchi, 1972).
2. Materials and Methods (a) Bacteria
and bacteriophage
coli strain B was used as the phage host. E. coli strain 1100, an endonuclease I defective mutant of K12 (Diirwald & Hoffman-Berling, 1968), was used for experiments Escher&h&z
in vitro. Bacteriophage 1963), were obtained Drake.
T4D and its ultraviolet from Dr W. Harm. T4p2
light-sensitive mutants, v1 and z (Harm, (Drake, 1973) was provided by Dr J. W.
(b) Media (i) M9 medium: NasHP0,.12H,O, 14.7 g; KH2P0,, 3 g; NaCl, 5 g; 0.1 M-MgS04, 10 ml; 0.01 M-CaCl,, 10 ml; 40% glucose, 10 ml in 1 1 of distilled water. (ii) Dilution fluid: M9 medium without glucose, CaCl, and MgSO,. (iii) Broth: polypeptone, 10 g; extract of fish meat, 5 g; NaCl, 3 g in 1 1 of distilled water. (c) Irradiation
and plating
of phages
Phages were suspended in dilution fluid and irradiated with a 15 W Toshiba germicidal lamp at room temperature. In most experiments, irradiation was at a distance of 80 cm of irradiated phage was from the lamp (approx. dose rate 0.6 J/ m2 per s). The plating performed in a dimly lit room to avoid photoreactivation. For control of temperature, plates were pm-incubated at desired temperatures and, after plating, placed quickly into an air-circulating incubator. In some experiments, plates were wrapped in a vinyl sheet and placed in a temperature-controlled water-bath incubator. (d) Isolation
of mutants
T4D wild-type
was mutagenized with 0.4 M-hydroxylamine in 0.05 M-sodium phosphate (Tessman, 1968). After incubation at 37°C for 48 h (survival of 10V2), the suspension was diluted and portions were added to a l-ml culture of E. coli B (1 x lo7 cells/ml) in broth and were incubated at 37°C overnight. In this step, mutants which usually occur in mixed clones segregate from the wild type. The lysates thus obtained were screened for u.v.-sensitive mutants by comparing survival after U.V. irradiation. To avoid selecting mutants originating from t,he same clone, a number of lysates were prepared and only one mutant was isolated from each lysate. The lysates were diluted and plated with E. coli B. Plaques wore picked up with capillaries which were then placed in 1 ml of dilution fluid. Three O.l-ml portions of the suspension were added to holes (6 mm diam.) in a plastic tray, and irradiated with U.V. light at various doses (0, 10 and 20 s at 80 cm from the lamp). By means of a sterile paper strip (5 mm x samples were applied to a plate, which had 70 mm) dipped into the phage suspensions, been seeded with about 10s cells of E. coli B in 3 ml of soft agar and pm-incubated at 42°C for 1 h. After administering phages tho plate was quickly placed in an air-circulating incubator at 42°C overnight. Approximate survival rates of phages were determined by the spot tests, and strains which exhibit increased U.V. sensitivity were selected and further tested for their U.V. sensitivities at 30°C and 42°C by an ordinary plaque-counting method.
buffer (pH 6*0), 0.001 M-EDTA
(e) Assay
of enzyme activities
Excision of pyrimidine dimers in vitro was determined as described by Sekiguchi et al. (1970). Sedimentation analysis of DNA in alkaline sucrose was performed as described previously (Yasuda & Sekiguchi, 19705).
~LTHAirlOLE’J’
LIGHT-YEXS11‘IYE: (f ) Preparatioll.
M[!1’A,L”1’S
OF ‘P-1
1;
of enzyme
E;. co/i 1100 was grown in broth at 30°C’ to a concentration of I x 10s cells/ml iufectrd with strains of T4 at, a mult’iplicity of infect’icln of 4. At 20 min after infection,
a~ltl the culture was chilled rapidly by pouring ont’o crushed ice. (-‘ells were collected by refrigerated cmt,rifugation, resuspended in a buffer containing 0.05 >r-Tris.HC’l (pH 7.5), 0.001 %I-?IYrrt)rokt~r1 cells and dnbris mercaptoethanol and disrupted in an ultrasonic disilltegrator. fraction was \vere rcmovod by crntrifugat,iotr at 10.000 g fol- 20 mill. antI ttle sli])ernatant Ilsed as a cell-frte rxtract.
A 1 -ml sample of bacterial culture exposed to 32P \vas \vit,hdraM.u and acidified iltmlc[l2 ml iately with trichloroacetic acid (final concn. 5”;). Thcr~ 0.1 mmol sodium phosphate. of an overnight culture of E. coli B and 2 x IO’O particlvs of T4D were added to the rnixturct as carriers. After extraction of acid-soluble materials, pllospbolipid and RNA, DNA was I !W). Tllr~ radioactivit>, of DNA uxs ext’ractod by hot trichloroacetjic acid (Sekigllcahi. determined in a gas-flow count,er.
3. Results (a) Isolution
of tenbpernt,ule-de~et~aet~,t
ultrar~iolet
light-.wtt,.sitice
mutanf.u
(1963) and Boyle t Symonds (1969) isolated u.v.-sensibivr mutants of 1’4 Harm with mutations in three different genes, V. z and y. We have tried t’o isolat’e a Ned type of u.v.-sensitive mutant by selecting mutants at high temperature. To ensur(a obtaining high frequency of mutation, T4 was mutagenized with hydrox.vlamine and u.v.-sensitive mutants were selected. Among 830 plaques examined, ten mutants were isolated ; five of them were temp~raturt,-dt,penderlt, u.r.-sensitive mutants. and the other five were ordinary u.v.-sensitive mutants. whose sensitivity was not significantly affected by temperature. Figure 1 shows survival curves of the tcmperature-dependent mutants and of the reference strains, T4D. and ‘vJ~.-it 42°C the mutants (F81. F431. F794. I?801 and F821) ~‘ere as sensitive as vI, but at 3O’C these mutants displayed a U.V. sensitivity identical to that of thr wild-type. The u.v. sensitivities of T4D and v1 are not significantly affected by t,emperature. When irradiated phages are plated at various temperat,uren. the mut,ants exhibit increased U.V. sensitiviby with elevating t’emperaturr and t hrh sharpest increase iI1 the sensitivity t,akes place between 34°C and 37,-C.
Among u.v.-sensitive T4 mutantSs. r and !/ arc’ sc&tivr to methyl methanesulfonate. whereas v is as resistant t’o t#he agent as t’he wild-type (Mortelmans & Friedberg. 1972; Ebisuzaki et ul., 1975). Thus. the mutants isolated were examined for their methyl methanesulfonate sensitivity. Phages were suspended at 1 x 1O8 plaque-forming units/ml in M9 medium containing 0.01 M-methyl methanesulfonatc and incubatted at’ 37°C. The susptbnsion was diluted at various times (20 to 60 min)
IS
Ii.
S:\‘I’O
.lNL)
\
I IO
\
I 20 Ultravlalet
(a)
Al. SEKIGU(‘HI
I IO
0 irradiation
I 20
time (s) (b)
FIG. 1. Ultraviolet light irradiation survtval CUWHS of T4 mutants. Phages (6 were irradiated at 80 cm from a lamp and plated with E. coli H on pro-warmed werr incubated at, 30°C (a) or 42°C (b). The same result was obtained when E. ashost, (-~-‘:-
