Photochernrriry and Phorohrology. Vol. 29. pp. 543 547. 0 Pcrgiimon P r w Ltd. 1979. Printed in Great Brit;tin

EVIDENCE FOR DARK REPAIR O F FAR ULTRAVIOLET

GLOEOCAPSA ALPICOLA LIGHT DAMAGE IN THE BLUE-GREEN ALGA,

EDWIN WILLIAMS, JOAN LAMBERT, PHILIP O’BRIEN and JAMES A. HOUGHTON Department of Microbiology, University College. Galway. Ireland

(Receiwd 16 June 1978; accepted I 1 Septetnher 1978)

Abstract-The inactivating effect of far U V light on the unicellular blue-green alga Gloeocupsu dpicolu could be totally reversed by exposure to blue light immediately after irradiation. However, if the irradiated cells were held in the dark before exposure to blue light, reversal became progressively less efficient and almost disappeared after 6&80 h holding. Caffeine and acriflavine inhibited loss of photoreversibility. suggesting an involvement of excision functions. Chloramphenicol and rifampicin slightly increased the rate of loss of photoreversibility, indicating that inducible functions play only a minor role. Split UV dose experiments indicated that light-dependent repair remained operational during dark liquid holding. These results provide preliminary evidence for dark repair in G. alpicolu.

INTRODUCTION

Cyclobutane pyrimidine dimers in the DNA of micro- organisms may account for more than 90% of the lethal and mutagenic consequences of irradiation by far ultra-violet light (FUV) at low fluences (Witkin, 1976; Doudney, 1976). The effects of FUV may be reversed or modified by the action of a variety of repair mechanisms (Swenson, 1976; Moseley and Wil- liams, 1977). Although these mechanisms overlap to a considerable extent they may be considered as pre- DNA replication or post-DNA replication events (Nishioka and Doudney, 1970). Pre-replication repair of FUV lesions occurs mainly by photoreactivation and excision repair (Doubleday et a/., 1975; Howard- Flanders et al., 1971) although the involvement of recombination and “SOS” functions has also been demonstrated (Cooper, 1977; Hanawalt et al., 1975).

Photoreactivation consists of the monomerisation of dimers in situ by a photoreactivating enzyme (PRE) which requires short wavelength visible light or near UV as an energy source (Harm, 1976). Excision repair is a multienzyme process with two main branches: a constitutive short patch branch and an inducible long patch branch (Cooper and Hanawalt, 1972; Youngs and Smith, 1973; Cooper, 1977). Short and long patch repair have a common initial step which involves the recognition of the dimer by a UV- endonuclease and cleavage at, or near, the site of damage (Grossman rt a/., 1975). Because the substrate for both the PRE and the UV-endonuclease is the cyclobutane pyrimidine dimer, competition for dimer sites occurs. Formation of a complex between PRE and a dimer prevents its cleavage by UV-endonuc- lease. Conversely, single strand nicks introduced close to the dimers by UV-endonuclease prevents the pho- toreactivation of the dimers (Patrick and Harm, 1973; Braun and Grossman, 1974; Grossman el al., 1975).

Harm ( I 968) investigated the competition between photoreactivation and excision repair by following P A P . 29 3-G 543

the loss of photoreversibility of FUV damage in E. coli B/r during dark liquid holding in buffer. He found that, among those lesions not dark repaired, the fraction of lethal lesions removed by photoreacti- vation decreased and virtually disappeared after 24-48 h holding. This effect was absent in the exci- sion-deficient E. coli strains B,-, , AB 2437 (uurA-6) and AB 2480 (uorA-6, rec-B), and was much reduced in E. coli B/r in the presence of caffeine and acrifla- vine, indicating that pre-replication excision events were responsible.

In the blue-green algae, considerable evidence for photoreactivation of FUV damage has accumulated (Delaney et al., 1976) and a partially purified PRE has been isolated and characterized (Saito and Wer- bin, 1970). However, no conclusive evidence of dark repair of FUV damage has been presented. In the present communication the work of Harm (1968) has been extended to the blue-green alga Gloeocapsu alpi- cola and preliminary evidence is presented for pre- replication dark repair events in this organism.

MATERIALS AYD METHODS

Orgunism. Gloeocapsu alpicola (Lyngbye) Bornet, a uni- cellular blue-green alga, was obtained from the Culture Collection of Algae and Protozoa, Cambridge. England (collection number 1430/1).

Media and growth conditions. The medium was a modifi- cation of that described by Allen (1968). Liquid growth medium contained, at final concentration (g// deionized distilled water): NaNO,, 1.50; K,HPO,. 3.9 x lo-*: MgS0,,7H2O, 7.5 x lo-’; CaCI,, 3.6 x lo-’; citric acid, 1.2 x lo-’; ferric citrate, 6.0 x lo-,; H,PO,, 2.9 x MnCI2, 1.8 x lo-,; ZnS0,.7Hz0, 2.22 x

Na2Mo04~2Hz0, 3.92 x CuS0,.5HZO; C O ( N O , ) ~ ~ ~ H ~ O , 5.0 x lo-’. Sodium silicate (0.58 g//) and EDTA (lO-’g//) were added and the mixture heated to boiling. After cooling, the pH was adjusted to 5.5 and the medium sterilized at 121*C for 15min. After cooling, filter. sterilized Na,CO, was added to a final concentration of 2.0 x to-’ g// giving a final pH of 8.c8.5. Flasks (500 m/)

544 EDWIN WILLIAMS e f trl .

containing 100 m/ medium were inoculated with single col- onies of G. ulpicolo or a log , inoculum of an exponential culture and incubated in a Gallenkamp illuminated, cooled orbital incubated at 30 1°C. 120 oscillations min- ' and under constant illumination with white fluorescent light at 2000Ix.

Solid medium was prepared by mixing sterile. double strength salts medium with an equal volume of sterile Difco Bacto agar (3.6% in distilled water) at 45°C. After rapid mixing the medium was poured into plastic petri dishes and the plates were dried at 60°C for IOmin.

Viable counts were determined by diluting liquid cul- tures of G. alpicolu in salts medium and 0.1 m/ aliquots of the appropriate dilutions were spread onto dried plates. The plates were incubated at 30 L- I'C, SO"/, relative humi- dity and illuminated with gold-yellow light (Thorn Inter- national Ltd. 1 550-700nm) at 2000Ix. Colonies were counted after incubation for I s 1 4 days.

For U V irradiation. Washed cell suspensions at a density of 108cells/m/ were exposed to 254nm FUV in petri dishes at room temperature. The source of FUV was a mercury discharge lamp (Griffin and George Ltd.) provid- ing an incident fluence rate of 0.2pJ min-* s-'. The sus- pensions were stirred during irradiation by a magnetic stirrer. Irradiations and all subsequent manipulations were carried out under yellow light to prevent photoreactiva- lion.

Photoreacrilwtion qf Far U V irrodiafed cultures. Two m/ samples of FUV-irradiated cultures were exposed, in sterile petri dishes. to blue light (Thorn International Ltd.. i. 350-550 nm) at an intensity of 2000 Ix. To ensure maxi- mum photoractivation. cultures were exposed to blue light for 90min.

Determinor ion ofloss ofphororeaersibilit!. ofFnr U Vdomaye during liquid dark holding. Exponential cultures were held in the dark at 30°C for 24 h before FUV irradiation to allow completion of all rounds of DNA replication. FUV- irradiated cultures were incubated in salts medium at 30°C in the dark and caffeine (0.2""). acriflavine (1 icg/m/), chlor-

amphenicol (150 pg/m/) and rifampicin (200 &m/) were added where indicated. Samples were removed at intervals. centrifuged to remove the repair inhibitors, resuspended in the original volume of growth medium and either plated directly or exposed to photoreactivating light before plat- ing for viable count determinations. Unirradiated control cultures were also liquid held in the dark for the same times and plated with or without exposure to photoreacti- vating light.

RESULTS

The survival of FUV-irradiated G. alpicola grown under yellow light (1 550-750 m) decreased exponen- tially after a small shouldered region at low FUV fluences (Fig. la). Exposure to 60min of blue light (1 35&550 nm) immediately after irradiation restored viability to pre-irradiation levels, suggesting the pres- ence of a mechanism for photorepair of lethal FUV lesions (Fig. 1 b). After a delay of approximately 4 h, the ability to photoreverse lethal FUV lesions was gradually lost during post-irradiation dark liquid holding (DLH) in non-growth conditions (Fig. 2a). Unirradiated control cultures and non-photoreacti- vated, FUV-irradiated cultures maintained constant viability during the holding period. A small fraction of the irradiated population was still photoreversible after long periods of DLH (Fig. 2a).

If loss of photoreversibility during DLH is an expression of excision functions, known inhibitors of excision repair, such as caffeine and acriflavine. should inhibit the process. DLH in the presence of caffeine or acriflavine considerably delayed the onset

0 P 40 60

Phobm&iuothgIight, min

Figure 1. Reversal of the lethal effects of far UV by immediate post-irradiation exposure to blue light. (a) (0) FUV-irradiated cells not exposed to blue light. (0) FUV-irradiated cells exposed to 90min blue light. (b) (0) Unirradiated control exposed to W m i n blue light. (0) Cells FUV-irradiated for

32 min and exposed for various times to blue light.

545

100

s - .- e z ' 3

01 *-•

- I0L 0 20 40 0 20 40

Post-UV dark holding, h

Figure 2. Effect of inhibitors of dark repair on the loss of photoreversibility of FUV damage during dark liquid holding. (a) Effect of 0.2% caffeine, (b) Effect of 1 pg/m/ acriflavine, (c) Effect of 150pg/m/ chloramphenicol. (d) Effect of 200 pg/m/ rifampicin, (0) Unirradiated cells exposed to 90 min blue light. (W) Unirradiated cells plus repair inhibitor exposed to 90 min blue light. (0) FUV-irradiated cells. (0) FUV-irradiated cells plus repair inhibitor. (Q) FUV-irradiated cells exposed to 90 min blue light after various periods of dark liquid holding. (*) FUV-irradiated cells exposed to 90min blue light after various periods of dark liquid holding in the presence of repair inhibitor. All irradiated

samples were exposed to FUV for 32 min.

of loss of photoreversibility and, in the case of caf- feine, slowed down the subsequent rate of loss (Figs. 2a, b). The fraction of the population retaining the ability to be photoreversed was increased by caffeine and acriflavine, and the survival of non-photoreacti- vated, FUV-irradiated cultures was decreased, pre- sumably as a result of inhibition of excision repair by these agents.

The photoreactivable sector was calculated by the method of Harm (1968). This is a measure of the maximum photoreactivation, defined as (1 - DI/D2), where D, is the dose that would give, without illu- mination, the same survival as does the FUV dose D, with maximum photoreactivation. The photoreac- tivable sector expresses the fraction of lethal FUV lesions abolished by photoreactivation, among those lesions that do not become dark repaired. The pho- toreactivable sector of FUV-irradiated G. alpicola de- creased during DLH but did not reach zero, even

after long periods of holding (Fig. 3). Both caffeine and acriflavine delayed the process by approximately 15 h.

To determine if inducible functions were involved in the process leading to loss of photoreversibility, chloramphenicol or rifampicin were added to the DL-held cultures. The onset of loss of photoreversibi- lity was not delayed by either chloramphenicol or rifampicin but the rate of loss was slightly increased by both compounds (Fig. 2c, d). The non-photoreacti- vated, FUV-irradiated cultures were, again, slightly sensi tized.

Although the results can be satisfactorily explained by competition between PRE and UV-endonuclease for FUV lesions, decay of PRE during DLH of FUV- irradiated cultures is an alternative explanation. This alternative hypothesis was eliminated by irradiating, for a second time, a DL-held culture showing maxi-. mum loss of photoreversibility. Exposure to blue light

546 EDWIN W ~ L L I A M S et trl.

0 40

Pad-UV darlc holding, h

Figure 3. Effect of caffeine and acriflavine on loss of pho- toreactivable sector during dark liquid holding of FUV- irradiated cells. (0) 1 pg/m/ acriflavine. (0) 0.2”,, caffeine.

(0) (D) FUV-irradiated controls: no repair inhibitors.

immediately afer the second FUV dose restored viabi- lity to that found after the first FUV irradiation. Dur- ing DLH, photoreversibility was again gradually lost (Fig. 4).

DISCUSSION

Photoreactivation describes the removal of the lethal and mutagenic consequences of FUV by post- irradiation exposure to short wavelength visible light or to near UV (wavelengths between 300 and 500 nm) (Harm, 1976). In E. coli, photoreactivation is mediated by a single enzyme which binds specifically to cyclobutane pyrimidine dimers which form part of a polynucleotide chain of at least nine bases (Patrick and Harm, 1973). Binding of the enzyme to the dimer and monomerization occur in the dark, but dissociation of the complex is light dependent (Harm. 1976).

Photoreversal of FUV damage has been demon- strated in a number of blue-green algae (Rupert, 1975) and a PRE with a mol wt of 93,000 and an absorption peak at 435 nm has been isolated from Anocystis nidu- luns (Saito and Werbin, 1970). PRE activity has also been demonstrated in extracts of Plactonerna bor- yonurn (Werbin and Rupert, 1968). Since photosyn- thesis in blue-green algae occurs efficiently in yellow light ( A 550-750 nm), whereas photoreactivation occurs at a negligible rate at these wavelengths (Rupert, 1975), the two processes can be effectively separated.

In G. alpicolu, exposure to blue light (A 350-550 nm) immediately after FUV-irradiation totally reverses the

lethal effects, suggesting the presence of a photoreacti- vation system analogous to that found in other organisms (Figs. la, b). However, if FUV-irradiated cells are liquid held in the dark before exposure to blue light the extent of reversal gradually decreases (Fig. 2a). During liquid-holding of UV-irradiated E. coli it has been demonstrated hat some steps of exci- sion repair may occur, resulting in strand incision, dimer excision and occasionally resynthesis and liga- tion (Tang and Patrick, 1977a). The first step of exci- sion repair is recognition of the dimer by a uurABC endonuclease which introduces a nick at, or near to, the site of the dimer (Grossman, 1975). PRE can bind normally to DNA nicked by the endonuclease, but the light-dependent step is inhibited. The nicked dimer, therefore, becomes non-photoreversible (Patrick and Harm. 1973). Loss of photoreversibility has been demonstrated in E. coli B/r and wild-type K12, but does not occur in strain A82437 (urrA-6). emphasizing the requirement for UV-endonuclease (Harm, 1968; Moss and Davies, 1974).

However, to account for the decrease in the photo- reactivable sector during liquid-holding, Harm (1968)

0 40 80

-.-I

0 20 60

Post-UV dark holding, h

Figure 4. Photoreversibility of a culture irradiated for a second time with FUV after showing maximum loss of photoreversibility from the first FUV dose. (D) Unirra- diated cells exposed to 90min blue light. (0) Cells FUV- irradiated for 20 min. (0) Cells FUV-irradiated for 20 min. dark liquid held for 100 h and again irradiated for 20 min. (*) Cells FUV-irradiated for 20 min and exposed to blue light for 90 min after various periods of dark liquid hold- ing. (*) Cells as (0) but exposed to blue light for 90min after various periods of dark liquid holding following the

second FUV-irradiation.

Dark repair in Gloeocupsu trlpicolu 547

proposed that the excision system occasionally pro- duces lethal lesions. Overlapping excision gaps may constitute such lesions and as the density of pyrimi- dine dimers increases, the chances of overlapping excision gaps in increased (Moss and Davies, 1974). Bonura and Smith (1975) have provided evidence that double strand gaps arise enzymatically during exci- sion repair of UV-irradiated wild-type E. coli K-12.

The involvement of excision functions in loss of photoreversbility of liquid-held G. ulpicola is sug- gested by the inhibition observed with the excision inhibitors, caffeine and acriflavine (Figs. 2a, b), and by the sensitization of non-photoreactivated, FUV- irradiated cultures. The marginal increase in the rate of loss of photoreversibility by rifampicin and chlor- amphenicol suggests that inducible functions may play a minor role (Figs. 2c, d). This is similar to exci-

sion repair in liquid-held E . coIi where the constitu- tive short-patch branch is dominant and the inducible long patch branch plays only a minor role (Cooper, 1977; Tang and Patrick, 1977b).

Loss of photoreversibility of FUV damage in G. alp ico la , therefore, provides preliminary evidence for the presence of a dark repair system, perhaps analo- gous to excision repair in E. coli. The alternative hy- pothesis, that PRE decays during DLH, has been eliminated by split-FUV-dose experiments (Fig. 4). Work is currently in progress to characterize these systems for repair in G. alp ico lu both biochemically and genetically.

Acknowledgements-This work was financed in part by Euratom under its Biology and Health Protection Pro- gramme, Contract No. 127174-1 BIO EIR.

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