MGG

Molec. gen. Genet. 146, 107- 115 (1976)

© by Springer-Verlag 1976

Genetic Exchanges Caused by Ultraviolet Photoproducts in Phage 2 DNA Molecules: The Role of DNA Replication Pin-Fang Lin and Paul Howard-Flanders Department of Molecular Biophysics and Biochemistry and Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

Summary. Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 (2) lysogens infected with genetically marked phage 2. Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed. First, samples of a heteroimmune phage 2 imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage 2 ci857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P+ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be attributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme. Second, in homoimmune crosses, samples of similarly treated homoimmune 2 P3 phages were allowed to infect lysogens carrying 2 ci857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by 2 immunity, and was further blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P+ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light. It is concluded from these results that certain uni-

dentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.

1. Introduction

The frequency of recombination in genetic crosses in various microorganisms can be increased by exposing them to ultraviolet light before mating. Ultraviolet-promoted recombination occurs to a greater extent in excision-defective mutants than in wild type cells, and it can be partially reversed by a subsequent treatment with visible light. Much of the increase may be due to pyrimidine dimers, as they are the only photoproducts known to be dissociated by photoreactivating enzyme (Jacob and Wollman, 1955; Wulff and Rupert, 1962; Setlow, Boling and Bollum, 1965; Howard-Flanders and Boyce, 1966; Baker and Haynes, 1967). This increase presumably reflects a greater frequency of pairing between strands of homologous molecules rather than any effect on the later steps of recombination. It has been proposed that the increased pairing might be due to: (1) the disruption of hydrogen bonding between complementary strands in the vicinity of pyrimidine dimers or other photoproducts (Curtiss, 1968; Baker and Haynes, 1972); (2) the formation of single-strand gaps with unpaired bases during the excision and repair of DNA containing photoproducts (Stacey, Evenchik and Hayes, 1969); or (3) to the replication of DNA and the formation of daughter strands containing gaps opposite pyrimidine dimers, the free strand ends atthe gaps being able to initiate recombination (Rupp

108

P.-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune ,~ Phage-prophage crosses

and Howard-Flanders, 1968; Howard-Flanders, Rupp, Wilkins and Cole, 1968; Wilkins and HowardFlanders, 1968; Rupp, Wilde, Reno and HowardFlanders, 1971). If the third hypothesis is correct, pyrimidine dimers should induce recombination only if the DNA containing them is replicated-a question that can be investigated in a bacteriophage ,~ system. Genetic recombination in phage 2 systems can be formed through the joining of fragments of preexisting or newly synthesized phage DNA molecules. Recombination may be associated with local DNA synthesis in the region of the join, but it can take place even when the replication of the phage DNA is repressed by 2 immunity or diminished by mutations affecting host DNA synthesis (Meselson, 1964; Stahl and Stahl, 1971). When lysogens are infected with virulent or heteroimmune phages, recombination between phage and prophage is greatly stimulated if the phage has been ultraviolet-irradiated. This recombination may contribute to the survival of the infecting phage (Jacob and Wollman, 1953 ; Yamamoto, 1967; Hart and Ellison, 1970; Blanco and Devoret, 1973). These studies indicate that genetic recombination can be induced by structural damage in phage DNA molecules, but give no insight into the role played by the replication of damaged DNA in the initiation of exchanges. Replication of the DNA containing pyrimidine dimers or other photoproducts may be required for the initiation of genetic exchanges (Rupp and Howard-Flanders, 1968). To investigate the suggested need for replication, we have studied recombination in phage 2 systems in which replication of the infecting phage containing photoproducts was controlled genetically. In these experiments, photoproducts were induced in the DNA of the phage 2 either by exposure to 254 nm ultraviolet light, or by irradiation with near ultraviolet light of wavelength 313 nm in the presence of photosensitizing agents related to acetophenone, which induce pyrimidine dimers and other products in DNA (Meistrich and Lamola, 1970). In crosses in which the damaged phage DNA did not undergo replication, the 254 nm photoproducts caused a marked increase in the frequency of genetic recombination. The effective photoproducts were not pyrimidine dimers since they were not susceptible to photoreactivation by visible light. The photoproducts induced by 313 nm light and acetophenone M were less effective per dimer in increasing the frequency of recombination. Contrasting results were obtained in a heteroimmune system in which replication of the damaged phage DNA was permitted. Both types of irradiation caused a marked increase in recombination. The effective photoproducts were susceptible to repair by exci-

sion and photoreactivation. Therefore, pyrimidine dim e r s appear to cause recombination only when the DNA in which they are contained undergoes replication. A preliminary note on this work has been published (Howard-Flanders and Lin, 1973).

2. Materials and Methods a) Bacteria and Bacteriophages The E. eoli K12 strains used are described in Table 1. The phages 2 c1857 1"80, and 2 c1857 1)3 were obtained from R.P. Boyce, ), imm434 PSO and 2 ci26 from R. Devoret, and 2 c171 from C. Radding. P3 and PSO are amber mutations and are suppressed by supE44 in strain CR34 (Bachmann, 1972).

b) Chemical and Media N-(m-acetylbenzyl)-N, N-dimethylethylene-diammonium dichloride (acetophenone D) was provided by A.A, Lamola, and fl-dimethyl-aminopropiophenonehydrochloride (acetophenone M) was obtained from Aldrich Chemical. Lambda buffer contains: 0.73 g of Tris-hydroxymethyl-aminomethane adjusted to pH=7.2, 2.46 g MgSO4.7 H20, and 0.05 g gelatin per liter of distilled water. Broth contains: 10 g Bacto tryptone, 5 g yeast extract, and 0.5 g NaC1 per liter of distilled water, adjusted to pH 7.0. Agar for 2 plates contains per liter of distilled water: 5 g Bacto tryptone, 8 g Bacto peptone, 1 g NaCI and 15 g Bacto agar. Luria plates contain: 10 g of tryptone, 5 g yeast extract, 0.5 g NaC1, 18.6 g agar and 0.5 g NaOH per liter of distilled water. Top agar contains: 6 g Bacto agar and 8 g NaCI per liter of distilled water. Scintillation fluid contains 42 ml Liquifluor (Packard) per 1,000 ml of toluene, and is used to count dry slips of chromatography paper.

c) Ultraviolet Light Source The source of 254 nm light was a 15 watt G.E. low pressure mercury germicidal lamp emitting radiation principally at 254 nm. Samples were usually exposed at 70 cm from the lamp at a dose rate of 1.0 w/m 2. The source of the 313 nm light was a medium pressure 500 W water~cooled mercury lamp (Phillips), with an interference filter (Ealing Corp.) which transmitted light of wavelength about 313 nm and filters 7-54 and 9-54 (Coming) to absorb light of shorter and longer wavelengths. The incident light intensity at 313 nm light was measured by potassium ferrioxalate actinometry (Calvert and Pitts, 1967).

d) Irradiation of 2 Phages in the Presence of Acetophenone Derivatives Aqueous solutions of acetophenone M at 0.07 M, or acetophenone D at 0.05 M, were diluted 0.05•5 ml of ), phage with titer about 4 x 109 pfu per ml in 0.01 M Tris-gelatin buffer. After holding for one to two hours at 37 °, the mixture was irradiated at 25 ° with 313 nm light in a layer up to 3 mm deep which transmitted 30% or more of the light. Correction was made for adsorption in calculating the doses.

P.-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune 2 Phage-prophage crosses

109

Table 1. Derivation and genetic characteristics of bacteria Strain number

Synonym

NH4485

W3110

NH4267

CR34

Prophage

-

Repair

Suppressor

Other characteristics (4)

Origin or reference

+

-

F - wild type

(1)

-

+

supE44

F - leu thr thy dra/m lac thi

(1)

NH4804

2 ci857 P3

+

supE44

F

Lysogen of CR34

NH4807

-

uvrA6

supE44

F - thy dra/m thi lac

Thr + Leu + His + US s recombinant of AB2437 (3) x CR34

NH4808

2 ci857 P3

uvrA6

supE44

F - thy dra/m lac thi

Lysogen of NH4807

NH4114

-

uvrA6

-

Hfr J2 thi

His + Met + UV s recombinant of Hfr J2 (1) xAB2437 (3)

NH4816

2 ci857 P80

+

-

wild type

Lysogen of W31 l0

NH4817

2 ci857 PSO

uvrA6

-

Hfr J2 thi

Lysogen of NH4114

NH4818

-

reeA1

supE44

F - dra/m thi lac str

Thy ÷ UV s recombinant of MA1079 Hfr fecAl (2) x Cr34

NH4819

2 ci857 P80

recA1

supE44

F - dra/m thi lae str

Lysogen of NH4818

NH4825

2 ci857 P3

recA1

supE44

F - dra/m thi lac str

Lysogen of NH4818

leu thr dra/m lac thi thyA

(1) Bachmann (1972) (2) MA1079 is a serA derivative (W. Maas) of KL1699, Low, 1968 (3) Howard-Flanders, Boyce and Theriot (1966). Note that AB2437 is a 2 lysogen (4) dra/m denotes a mutation in dra or drm which blocks the utilization of thymine as a carbon source and so permits growth in low concentrations of thymine

e) Homoimmune Crosses between Phages and Prophages in 2 Lysogens Log phage E. coli lysogenic for 2 ci857 P80 was grown at 30 ° to 2 x 108 cells per ml, and samples were infected with experimentally treated phage in broth or in 2 buffer to give a multiplicity of about 5 phage 2 ci857 P3 or 2 cI P3 per cell and incubated for 30 minutes at room temperature. The samples of infected cells were diluted 1,000-fold with broth and incubated overnight without aeration at 30 °. The following day, the number of lysogens per ml in each culture was measured by plating them on Luria agar and counting the number of colonies formed after incubation overnight at 30 °. The number of lysogens carrying P+ recombinant prophages was determined separately by plating them in soft agar on sup+(su -) indicator bacteria (NH4485), incubating at 42 ° overnight, and counting the number of plaques. The best plaques were obtained with 2 ci857 lysogens by incubating at 42 ° for 2.5 h and then at 37 ° overnight. f ) Heteroimmune Crosses between 2 imm434 Phages and 2 Prophages in Lysogens Carrying supE44 The supE44 bacteria lysogenic for 2 ci857 P3 were grown in broth at 32 ° to 2 x 108 cells/ml, infected with the experimentally treated 2 imm434 P80 cI at a multiplicity of 1 to 5 phages per bacterium, spun, and then resuspended in broth to remove the unabsorbed phages. The infected lysogens were then diluted to about 2 x 106 celis/ml in broth and incubated with aeration at 32 ° for 2.5 h. After shaking with chloroform, the suspension was assayed for P+ and total phages by plating on sup + (NH4485) and supE44 (NH4267) indicator bacteria.

at 2 x l08 per ml were infected with the irradiated phages at a multiplicity of about 1 and immediately exposed at about 5 cm below two 40 w white fluorescent lamps for 30 min at 25-30 °. The light of wavelength less than 300 nm was absorbed by a 1 cm glass plate. After exposure, the infected cells were spun at 6 krpm for 10 rain to remove the unabsorbed phage, diluted and grown as described.

h) Preparation of Radioactive Phage 3H-)~ ci26 or 2 cI71 was prepared by infecting wild type E. coli K12 (NH4485) at a density of 4 x 10 s cells/ml in K medium supplemented with 20 m M MgSO 4, and 100 pg/ml deoxyadenosine at a multiplicity of infection of about 2. After 15 min growth at 37 °, 4.5 mCi aH-thymidine was added to each liter of culture. Growth was continued until lysis had occurred at about 2.5 h. The lysate was treated with chloroform and bacterial debris was removed by centrifugation at 6 krpm 10 rain, giving a titer of 34 x 1010 pfu/ml. To one liter of culture, 1 mg of pancreatic deoxyribonuclease and 1 mg of ribonuclease were added and incubated at 37 ° for 30 min with slow shaking. The phage stock was concentrated by centrifugation at 23 krpm for 60 min in a Spinco 30 rotor. After discarding the supernatants, 1 ml of 2 buffer was added to each pellet and allowed to stand overnight. Following a second low-speed centrifugation, the phages were dialyzed overnight against cold 2 buffer. The purified stocks had a titer of about 4 x 1011 pfu/ml, and contained about 2 x l0 s 3H cpm/ml.

i) Chromatography of Pyrimidine Dimers g) Photoreaetivation To test the susceptibility of UV-induced recombination to photoreactivation by visible and near ultraviolet light, the lysogens

all-2 phage, at about 4 x 1011 pfu/ml and 2 x 105 cpm/ml, were • exposed to 313 nm light in the presence of acetophenone M or acetophenone D, or to 254 nm light alone. The sample thickness

110

P,-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune 2 Phage-prophage crosses

ure 1A. The chromatographic mobility of peak I (Rf=0.24) was similar to the value 0.19 to 0.22 reported for UT, while the Rf value of peak II (0.32.) is similar to the value 0.24 to 0.31 reported for TT and the adduct U-T (Smith, 1963 ; Setlow and Carrier, 1966; Varghese and Wang, 1967; Wang and Varghese, 1967). The chromatograms of photoproducts produced in the phage DNA by irradiation in the presence of acetophenone D and M are shown in Figures 1 B and 1 C. These chromatograms show the peak II with Rf value 0.33 attributed to TT, and the relatively small peak I attributed to ~ dimer. These results confirm that relatively small yields of C~ dimers are obtained in sensitized irradiations (Meistrich and Lamola, 1972). The results are summarized in Table 2. In the case of 254 nm light, a dose of 10 J/m 2 produced 3.6 pyrimidine dimers per phage (c.f. 4 dimers per phage: Radman, et al., 1970), or 78 pyrimidine dimers per million base pairs (c.f. 130 pyrimidine dimers per million base pairs in E. eoli (Rupp and Howard-Flanders, 1968). The corresponding yields for exposure to 313 nm light in the presence of acetophenone D and acetophenone M are 0.15 and 0.3 pyrimidine dimers per million base pairs respectively in phage 2. These results enable the effects of the 254 nm UV light and the sensitized irradiation with 313 nm light to be compared on the basis of exposures producing the same number of pyrimidine dimers in the phage 2 DNA.

was less than 2 mm, and the effective UV dose was calculated allowing for adsorption. The acetophenone was removed by dialysis overnight against cold 2 buffer. To test for photoproducts in the phage DNA, the treated phages suspended in 2 buffer were brought to 0.3 N NaOH, held at 37° for 2 h, and then neutralized with hydrochloric acid. One hundred twenty-five Ixg of calf thymus DNA in solution was added to each ml of phage as carrier, and the DNA was precipitated by 5% TCA at 0°. After 30 min, the suspension was centrifuged at 9 krpm for 15 min. The pellet was washed with 5% TCA and then dried in an evacuated dessicator. The use of alkali was needed when chromatographing DNA from phages treated with acetophenone. The pellets were then hydrolyzed in 0.75 ml of trifluoroacetic acid at 170° for 90 rain. The hydrolysates were dried, taken up in 50 I11 of water, and subjected to descending paper chromatography on Whatman number 1 filter paper in n-butanol, acetic acid and water (80:12:30) for 16 h. The paper was cut into 6 mm wide strips, placed in 2 ml of toluene scintillation fluid and counted in a Packard scintillation counter.

3. Results

a) Yields of Pyrimidine Dimers in the DNA of Phage 2 Irradiated with 313 nm Light in the Presence of Acetophenone Derivatives or Irradiated with 254 nm Light Alone The yields of thymine dimers (TT) and cytosine thymine dimers (C'T) in phage 2 DNA were measured after irradiation with 254 nm light. Phage 2 ci26 or 2 eI71 labeled with 3H-thymidine, was irradiated, hydrolyzed in acid and analyzed by chromatography as described. The chromatogram for the photoproducts induced by 254 nm light are shown in Fig-

80

A

x 1/100

80

C

B

h5

300

ACETOPHENONEM 7,100 d/m 2 515 nm LIGHT

60

W 60 I--

170 d/m 2 254 nm

Z n.W 0.. 4O

ACETOPHENONE D

137~0~hOg ] T JI/GHT

1I

t,Z 0

,l

"l

200

÷

!I~, ~

'

/II

2O

'

~,

' ÷I. 'XCONTROL \ If X] 0.24 0.52

] 0.5 Rf

N

bt

" ~ t~~ ; * I '1.0 0

]l

0.33

CONTROL

6~\ I; 'A" 0

o

13a~B .=1%

I

I

0.5

1.0

Rf

Po !

CI!TROL 0.25

0.5

1,0

Rf

Fig. 1A-C. Distribution of radioactivity in paper chromatograms of acid hydrolysates of irradiated 2 phages labeled with 3H-thymidine. A 170 J/m 2 254nm light. B 10x 103 J/m 2 313 nm light delivered in the presence of acetophenone D. C 7.1 x 103 J/m z 313 nm light delivered in the presence of acetophenone M. The chromatograms were developed in n-butanol: acetic acid: water (80:12:30). The chromatographic mobility of peak I (Rf=0.24) is similar to that of uracil-thymine dimers, and the Rf value of peak II (0.32) was similar to that of thymine-thymine dimers and uracil-thymine adducts. The main peak at Rf=0.62 is thymine

P.-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune ). Phage-prophage crosses

111

Table 2. Pyrimidine dimers in the D N A of phage 2 c26 or 2 c71 exposed to 254 nm light alone, or to 313 nm light in the presence of acetophenone D and acetophenone M Irradiation Wavelength (rim)

Dose (J/m 2) (1)

Acetophenone sensitizer

Phage survival (%) (2)

Pyrimidine Dimers Fraction of Number of 3H-T in TT P ~ y per and T ~ phage D N A

Figure

(%) (3) 254 254 254 254

73 73 170 170

-

313 313 313 313

5,900 9,800 9,500 10,000

D D D D

313 313

4,000 7,100

M M

24 31 3 3

0.24 0.19 0.45 0.37

38 26 56 54

4.7 0.05 1.3 0.17

0.32 0.65 0.37 0.46

37 76 43 53

1B

3.6 0.026

0.43 0.69

50 80

1C

1A

(1) The mean dose average throughout the irradiated sample (2) The percent survival of the plaque-forming capacity of the phages (3) The percentage of the radioactivity from 3H-thymidine found in the photoproduct peaks I (~"T) and II (T'T). To calculate the numbers of pyrimidine dimers produced, the radioactivity in peak II was halved since each dimer contained two labeled thymines. The number of thymine residues per phage was taken to be 2.3 x 104

b) Recombination Caused by Photoproducts in )~ Phage DNA Undergoing Replication during the Lytic Infection of E. coli K12 ,~ Lysogens in Heteroimmune Crosses

during lytic infection, the heteroimmune phage 2 imm434 cI P80 was irradiated as described, allowed to infect E. coli supE44 (2 ci857 P3) at a multiplicity of infection of 1 to 5, and incubated with aeration at 30 ° for about 2.5 h. After lysis, the progeny phages were scored for P+ and P - by plating on indicator bacteria carrying supE44 and supE +. The reversion

To determine the extent of the genetic recombination induced by photoproducts in bacteriophage 2 DNA

ACETOPHENONE M 8~ :313 nm L I G H T

2 5 4 nm LIGHT

10 9

A

moi = I

B

2 5 4 nm L I G H T

moi = I

C

moi = 5

WT

m

E Q: i 0 8

10 8

hi Dhi (9

~

"~+VISIBLEWT

=< i 0 7

l0 7

n

+VISIBLE

.J

Fig. 2A-C. The yields of P+ recombinant and total 2 phages from heteroimmune crosses is plotted as a function of the ultraviolet dose delivered to the phages before infection. E. coli K12 supE44 (2'cI857 P3) lysogens were infected with ultraviolet-treated 2 imm434 P80 cI at a multiplicity of about 1 A, B or about 5 C. The infected lysogens were allowed to grow and the phages released assayed by plating on supE ÷ (NH4485) and supE44 (NH4267) indicator bacteria. The lysogens used were: NH4804 (wild type), NH4808 (uvrA6) and NH4825 (recA1). Also shown on the lower scale are the numbers of pyrimidine dimers per phage as determined by chromatography

~ "Z "K LIBHT

F-

o

10 9

IO 6

E " w,,, IOft ,,, o_ (9 "I" ~ iO 5 a. z + O ,o I0 4 no

I

I

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106

105

rec

,

,

50

I

I00

150

0

2000

J/m 2

0

4000

50

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iO 4 I00

150

d/m 2

[

I

t

I

I

L

25

50

0

25

50

o

DIMERS

PER PHAGE

PYRIMIDINE

A

,

20

112

P.-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune 2 Phage-prophage crosses

A/

ACETOPHENONE M 8~313nmLIGHT

254nrn LIGHT uvrA

a ~

uvrA

3 Z

_z

ee~

/ / +VISIBLE ;GH,~/I'0uVrA

2

O ¢3 lal [-Z W

ll/~ WT

1

/

~

I

~._~-oWT

50 100 150

uvrA

0

2500

5000

7500

dlm z I

I

I

I

I

25

50

0

25

50

PYRIMIDINE DIMERS PER PHAGE Fig. 3A and B. The fraction of phage 2 P+ recombinants in the total phage yield from the heteroimmune crosses of Figure 2A and 2B. The fraction of recombinants is plotted as a function of the ultraviolet dose delivered to the infecting phages, the numbers of pyrimidine dimers per phage being shown on the lower scale. The increase in the frequency of recombinants is greatest in the strain carrying uvrA. Exposure of the infected lysogens to visible light reverses the effect of irradiation, indicating that most of the effective photoproducts are photoreactivable and are therefore presumably pyrimidine dimers. A 254 nm light. B 313 nm light with acetophenone M

rates of the amber mutations were not increased by irradiation and remained below 10 .6 in crosses between irradiated phages carrying identical mutant alleles of P. The results from these heteroimmune crosses are shown in Figures 2 and 3. The total number of phages produced in crosses in wild type cells at a multiplicity of 5 remained almost constant for ultraviolet doses up to 150 J/m 2 (Fig. 2C), but fell ten-fold at a multiplicity of 1 (Fig. 2A, 2B). This treatment reduced the survival of phage to 2% on wild type and to less than 10 - 3 o n excision-defective hosts. As shown in Figure 3, the fraction of phages that were P+ recombinants increased many-fold after irradiation with 254 nm light. This increase was dependent upon recA + (Fig. 2C), and was greater in uvrA- (Fig. 3) and uvrC- (not shown) than in wild type lysogens, indicating that the recombination is caused by unexcised photoproducts, and that excision is not a prerequisite for this type of induced recombination. The level of recombination was substantially reduced in lysogens after treatment with white light (Fig. 3). Insofar as photoreactivation is specific for pyrimidine dimers, this result indicates that dimers are the photoproducts responsible for most of the observed increase in the frequency of genetic recombination.

Similar results were obtained in heteroimmune crosses when the infecting phages were treated with acetophenone and 313 nm light. As shown in Figures 2 B and 3 B, the yield of recombinants increased in proportion to the number of thymine dimers per phage as before. Similar results were obtained in experiments with acetophenone D (data not shown). Again, more genetic exchanges were detected in uvrA - than in wild type lysogens, while fewer exchanges were detected if the infected cells were photoreactivated by an exposure to white light (data not shown). Evidently, the phage treated with acetophenone and 313 nm light are still able to inject their DNA into the lysogens and to initiate lytic infection efficiently. As shown in Figures 3A and B, the effects upon the frequencies of recombination are similar tO those of 254 nm UV light.

c) Recombination Caused by Photoproducts in the D N A o f Repressed Phages in Homoimmune Crosses in which the Replication o f the Phage D N A is Minimized

The next experiments were designed to investigate recombination induced by photoproducts in the DNA of nonreplicating 2 phages. Phage 2 ci857 P3 was exposed to 254 nm UV light, or to 313 nm light in the presence of acetophenone derivatives, to induce pyrimidine dimers and other photoproducts in the phage DNA. The treated phages were allowed to infect E. coli K12 supE ÷ lysogens carrying prophage ,~ ci857 P80 at a multiplicity of 1-2. Note that the supE + cells are nonpermissive for P3 and P80 mutations. After growth through ten or more generations at 30°, the lysogens (by then virtually free of infecting phages) were tested for the fraction carrying P+ recombinant prophages. During growth at 30°, the replication of the infecting phage DNA was repressed by 2 immunity as well as being blocked by the lack of P+ gene product, which is needed for the initiation of phage DNA replication (Ogawa and Tomizawa, 1968). In these crosses, the total number of lysogens was determined from the number of colonies formed on complete agar at 30°, while the number of lysogens carrying P+ recombinant prophages was determined from the number of infective centers formed on indicator cells carrying supE + at 40 ° to 42 °. As seen in Figure 4A, the exposure of the infecting phages to 254 nm light causes more recombination in wild type than in uvrA lysogens. The effect of exposing the infected lysogens to visible light is small, and is to increase rather than decrease the frequency of P-- recombinants. This result differs from that seen in Figure 3 for heteroimmune crosses. Photoreactible pyrimidine dimers do not appear to induce recombi-

P.-F. Lin and P. Howard-Flanders: Ultraviolet-induced Recombination in Homoimmune 2 Phage-prophage crosses 0,8 ACETOPHENONE 3 1 3 nm LIGHT

2 5 4 nm LIGHT 0.6

A

+VISIBLE -'~ LIGHT ,~

B

03 0.4 Z

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