289

Mutation Research, DNA Repair, 254 (1991) 289-298 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100073K

MUTDNA 06436

DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: Genetic analysis J. H o l l a n d a, I.B. H o l l a n d b a n d S.I. A h m a d

a

a Department of Life Sciences, Nottingham Polytechnic, Clifton Lane, Nottingham N G l l 8NS (Great Britain) and b Institute of Genetics and Microbiology, Batiment 409, Unioersity of Paris XI, 91405 Orsay, Cedex 05 (France)

(Received 27 July 1990) (Revision received 5 December 1990) (Accepted 14 December 1990)

Keywords: 8-Methoxypsoralen, DNA damage by; Hyper-resistant phenotype, E. coli; PUVA, 8-methoxypsoralen

Summary Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied. Results show that a mutation, located at 57.2 min on the linkage map of E. coli, is responsible for the hyper-resistant phenotype. It is also responsible for the synthesis of a 55-kdal protein in high concentrations. In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C. There are indications that the mutation may have occurred in a regulatory gene, puuR, and as a result the operon, including a putative puoA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively. A model for the control of the PUV operon is proposed.

PUVA (a combination of 8-methoxypsoralen and ultraviolet light of 320-400 urn) is a chemotherapeutic agent for psoriasis and mycosis fungoids (Scott et al., 1976). There are a number of target sites in cells for this agent including DNA, cellular proteins, lipids and membranes (Midden, 1988). Of these the most significant site is DNA. 8-Methoxypsoralen (MOP) is a naturally occurring heterocyclic compound having two photochemically reactive sites; a furan end and a pyrone end. The reaction with MOP and DNA takes

Correspondence: Dr. S.I. Ahraad, Department of Life Sciences, Nottingham Polytechnic,Clfton Lane, Nottingham, NGll 8NS (Great Britain).

place in three stages. In stage one a molecule of MOP intercalates non-covalently between two bases in the complementary strands of DNA. In stage two a covalent monoadduct is produced by a photon from NUV. Subsequently when exposed to a second photon of N U V a proportion of the bound psoralen molecules form a covalently bound adduct with another pyrimidine base in the complementary strand of DNA (Cimino et al., 1985; Smith, 1987). MOP reacts mainly with thymine bases to form thymine psoralen adducts or thymine psoralen thymine crosslinks (Dall'Acqua, 1977). However, photoadducts to cytosine and purines have also been reported (Davies, 1980; Calvin and Hanawalt, 1987). It is presumed that intercalation of MOP into DNA causes significant distortion in the helix. Although the crosslinks are of greater

290 biological significance than the monoadducts, both are substrates for DNA repair enzymes (Cole et al., 1976). The repair of PUVA-induced D N A damage has been studied in phages, T3 and T4 (Strike et al., 1981; Belogurov and Zavilgelsky, 1981), in Escherichia coli (Sinden and Cole, 1978; Cupido and Bridges, 1985), in D. radiodurans (Kitayama et al., 1983), in yeast (Chanet et al., 1985) and in mammalian cells (Zolen et al., 1984). In E. coli, PUVA-induced DNA damage is repaired by complex repair mechanisms and the exact process is not yet clear. Genetical and biochemical studies of E. coli identified several proteins necessary for the removal of psoralen crosslinks. They include uvrABC, uvrD (helicase II), recA gene products and polymerase and 5'-exonuclease activities of polA (Cole, 1973; Cole et al., 1976; Saffran and Cantor, 1984). The uvrABC gene products are involved in the repair of both monoadducts and crosslinks (Cole, 1973). For monoadducts it has been proposed that the uvr excision nuclease (uvrABC gene products) makes cuts in the DNA strand, one on each side of the damage. The single-strand gap produced, as a result of the removal of monoadducts, is resynthesised by D N A polymerase-I (Sancar et al., 1985; Seeberg, 1981; Van Houten et al., 1986). In some cases however the incision gap may be enlarged by the 5' --* 3' exonuclease activity of Pol-I, possibly aided by the uvrD gene product and helicase II (Kumura et al., 1983). The enlarged gap may be subject to the long patch excision repair (Hanawalt et al., 1981). In an attempt to determine the precise role of the uvrABC excinuclease in D N A crosslink repair, Van Houten et al. (1986a) prepared a synthetic D N A fragment of 40 base pairs containing a psoralen crosslink at a unique position. The D N A was treated with uvrABC excinuclease and the resulting product analysed. It was found that the enzyme incised the 9th phosphodiester bond 5' and the 3rd phosphodiester bond 3' to the thymine residue attached to the furan ring of the psoralen. The strand having the pyrone ring of psoralen was not incised. Working with 4,5',8-trimethylpsoralen, Jones and Yeung (1990) have shown that the DNA-base composition determines which of the two crosslinked D N A strands will be incised by

uorABC excinuclease. GC enrichment of the region 6-12 bases 5' of the modified T on the furan side strand results in preferential incision of the furan side strand. When the G C rich region is on the 3' side or on neither side, incision occurs on either strand. In an attempt to analyse further the repair pathway for PUVA damage in E. coli, mutants hyper-resistant (SA270) and sensitive ( J H l l l ) to PUVA have been isolated and analysed. Preliminary studies with the mutant bacteria, SA270, have shown previously that a protein of approximately 55 kdal is synthesised in higher concentrations than in its wild-type parent strain (Ahmad and Holland, 1985). We have studied these mutants of E. co# in more detail in order to understand the DNA-repair mechanisms of PUVA-induced damage and investigate the regulation and possible role of the 55-kdal protein in such repair processes. Materials and methods

Bacterial strains The bacterial strains used in this study are listed in Table 1. Materials Near-ultraviolet irradiation was carried out using Sylvania Fluorescent Lamps (4 x ) emitting black light and having a peak at 366 nm. Far-ultraviolet irradiation was carried out using a Phillips ultraviolet light (TUV 15W G15 T8) having a peak at 254 nm. 8-Methoxypsoralen, mitomycin C, nitrogen mustard and ethyl methanesulphonic acid were all purchased from Sigma (London). LB medium (pH 7.5) contained Bacto-tryptone, 10 g; Bacto yeast extract, 5 g; sodium chloride 10 g; water 1 litre. Oxoid nutrient agar and nutrient broth were used routinely. Phosphate buffer (K2HPO4, 0.1 M and KH2PO4, 0.1 M; pH 7.0) was used for resuspending cells and for serial dilutions. M9 medium was prepared according to Clowes and Hayes (1968). Cell lysis Cell suspension was disrupted by either ultrasonication (Soniprep 150, MSE Scientific Instruments, Sussex) for a total of 2 min (20 sec bursts;

291 TABLE 1 Strain ( E. coli) KL16 Hfr SA270

Relevant genotype Prototroph puoR

SA315

recA::TnlO

SA316

recA::TnlOpuvR

JC13013

recJ 153

JC7623

recBC sbcB

SA256 JC9239

recB C recF 143

JC8111

recBC sbcB recF

SP254

recN 262

SA162 X121

uvrA thi pro tyrA

JH108

tyrA nal-r

JHlll

puvRpuoA::lacZTn5 kan-r

JHll0 KL711

puvRpuoA::laczTn5 kan-r F'143, pyrD, trp45, his 68 tyrA2, thyA

Source Bachmann (1972) Ahmad and Holland (1985) Ahmad and Holland (1985) Ahmad and Holland (1985) Lovett and Clark (1984) Kushner et al. (1971) From Bachmann Horii and Clark (1973) From Bachmann Picksley and Lloyd (1984) This laboratory From Bachmann nal-r derivative of X121

broth to a titre of 1 x 10 8 cells/ml. Cells were harvested and resuspended in 10 ml of phosphate buffer ( p H 7.0). The cells were lysed using a French Pressure Cell and SDS-polyacrylamide gel (10% v / v ) electrophoresis of the total protein extracts was carried out according to Laemmli (1970). P1 transduction mapping P1 transduction experiments were carried out according to A h m a d and Pritchard (1969). Measurement of bacterial survival against various DNA-damaging agents Bacteria were grown at 37°C in LB broth to approximately 2 x 10 8 cells/ml. A 1-ml aliquot of cells was added to 9 ml of phosphate buffer (pH 7.0) containing mitomycin C or nitrogen mustard, each at 5 0 / L g / m l concentrations. Samples were removed at known time intervals, serially diluted and plated on nutrient agar plates. Colonies of survivors were scored after 24 h incubation at 37°C.

This laboratory This laboratory Brooks Low (1972)

6 # m peak to peak at 0 - 5 ° C ) or by repeated passage ( x 2 ) through a French Pressure Cell (Moore Max-Ton Pressure, Birmingham, England) at 10 ton/sq, inch. Measurement of cell survival after P U V A treatment A 1-ml aliquot of an overnight culture in nutrient broth was added to 9 ml of phosphate buffer ( p H 7.0) containing 25 # g / m l , 8-methoxypsoralen. The culture, in a glass petri dish, was exposed under the near-UV lamp for a required dose, samples removed at known time intervals, serially diluted and plated on nutrient agar plates. The number of colonies appearing after 24 h incubation at 37°C were recorded. Polyacrylamide gel electrophoresis of total cell lysates Extracts fo r polyacrylamide gel electrophoresis were prepared by growing cells in 200 ml of LB

Induction of 55-kdal protein Overnight grown cultures in M9 medium, supplemented with kanamycin (30 # g / m l ) , were subcultured into fresh similar medium. When the cultures reached to exponential phase of growth (OD600 = 0.2) they were centrifuged and pellets resuspended in equal volumes of fresh M9 medium. Cultures were then either exposed to PUVA, or MC added to the cultures. Treated cultures were then incubated for the desired period at 37°C and samples removed at intervals. Subsequent treatment of cells and measurement of fl-galactosidase activities were essentially carried out according to Miller (1972). Units of activities are expressed according to the formula provided. Results Sensitivity of SA270 for DNA-damaging agents In their studies A h m a d and Holland (1985) tested the sensitivity of strain SA270 for PUVA and for far-UV. SA270 was found to be more resistant to P U V A than the wild-type strain, KL16. Both SA270 and KL16 were equally sensitive to far-UV. In this study SA270 and its wild-type

292 100

100

•

SA270

•

KL16

SA270 KL16

cn

o

>

10

10 c 0 o 0 a.

0

5

10

15

Time (mins) of exposure to nitrogen mustard (50pg/ml).

Fig. 2. Bacterial kill curve of KL16 and SA270 in the presence of nitrogen mustard.

1 0

5

10

15

Time (rains) of exposure to mitomyein C ( 5 0 p g / m l ) ,

Fig. 1. Bacterial kill curve of KLI6 and SA270 in the presence of mitomycin C.

parent strain were tested against other DNAdamaging agents. The results showed that the mutant strain is also hyper-resistant to the crosslinking agents MC and nitrogen mustard (NM) (Figs. 1 and 2). Analysis of SA270 for sensitivity to monofunctional DNA-damaging agents such as nitrosoguanidine and ethyl methanesulphonic acid showed that it remained as sensitive as its parent strain, KL16 (data not presented). Genetic mapping of the purR locus Preliminary conjugation experiments using SA270 as an Hfr donor and an F multiple auxotrophic mutant as a recipient showed that the gene responsible for the PUVA hyper-resistant phenotype (designated p u r R - ) is located near the recA gene at 58 min on the linkage map of E. coli (Bachmann, 1990). For fine genetic mapping, P1 transduction experiments were employed. SA270 was used as a donor and a tyrA-, nalB-r mutant (JH108) as recipient. Data (Table 2) show that the

co-transduction frequency between tyrA and nalB is 6% and between tyrA and purR is 41%. From the lack of puv + nal-s class of recombinants (requiring 4 crossovers) it was concluded that the p u r r gene is located between nalB and tyrA. Using the formula of Wu (1966) the exact position of the purR gene on the chromosome was calculated at 57.2 min. The genetic order therefore is tyrA - - p u r r - - nalB. To confirm the mutation located at 57.2 min was solely responsible for the PUVA hyper-resistant phenotype and for the overexpression of the 55-kdal protein, a tyrA + p u r R - transductant was analysed for the presence of the 55-kdal protein in a total cell lysate using SDS-PAGE. The results (Fig. 3) showed that the protein was overexpressed in the tyrA + p u r r transductant suggesting a link between the purR mutation, the PUVA resistant phenotype and the overexpression of the 55-kdal protein.

TABLE 2 T R A N S D U C T I O N A L DATA Donor

Recipient

tyr +

nal-r purR

nal-s puvR

nal-r puv + b

nal-s puv +

SA270

JH108

129

46

7

76

0

puvR, PUVA hyper-resistant cells. b puv +, Resistant to PUVA as a normal wild-type cell. a

293

Complementation analysis of the puvR locus To determine if the mutation at 57.2 rain is present in a regulatory gene (for example, equivalent to the lacI-), a complementation analysis, using an F' was carried out. The F' carrying the tyrA to iysA region of the chromosome, from strain KL711, was transferred by conjugation to the F - puvR- tyrA- strain, JH109. Selection was made for colonies able to grow on minimal agar plates lacking tyrosine. 20 trans-conjugants were screened for PUVA. 17 of them were as resistant to PUVA as the wild-type KL711 (result of one such ex-conjugant is given in Fig. 4) and 3 were hyper-resistant to this agent. Subsequent analysis of these derivatives showed that the 3 trans-conjugants, hyper-resistant to PUVA, were tyrosine auxotrophs suggesting that they had lost the F'.

200

92.5

69

m55 45

30

a

b

Fig. 3. Polyacrylamidegel electrophoresisof total cell protein prepared as describedin Materialsand Methods(a) wild-type, KL16(b) puoR- derivativeof KL16.

The others were prototrophic for tyrosine and presumably retained the F'. Since the results show that in puo +/puoR- merodiploid the cells were of wild-type phenotype it implies that the purR + gene is trans dominant over purR-.

Induction of 55-kdal protein To determine if the synthesis of the 55-kdal protein is inducible, cultures of wild-type E. coli KL16 were first grown with MC (2/~g/ml for 30 min) or treated with PUVA (20/~g/ml psoralen + 200 J//m2 NUV) and subsequently grown for 30 min. Treated cells were lysed by French Pressure Cell and the total cell extracts were analysed by SDS-PAGE. No difference in the profiles of the two extracts, with or without inducers, could be detected. One possible reason for this is that the method is not sensitive enough to detect the difference between the induced and the basal level synthesis of this protein. Hence a novel and more sensitive approach was taken. For this, an E. coil strain was first produced in which lacZ gene was fused, in frame, into the putative puvA gene (structural gene for the 55-kdal protein). Thus, instead of measuring the levels of 55-kdal protein the induced (and basal level) synthesis of the protein was estimated via the activity of fl-galactosidase. The construction of the strain involved SA270 (Synthesising 55-kdal protein constitutively) and this was lysogenised by PI:: Tn5 lacZ kan-r (Kroos and Kaiser, 1984). Lysogens were plated on McConkey's agar plates supplemented with kanamycin (25 /tg/ml) and cells producing deep blue colour colonies after 24-h incubation were analysed for PUVA sensitivity. An isolate (JH110) showed desired characters; it was sensitive to PUVA, produced fl-galactosidase constitutively and the PUVA sensitivity mutation mapped near the purR locus close to tyrA (data not presented). This strain was considered to carry Tn5 lacZ in the putative puoA gene. Genotypically, therefore, it is p u r R - puvA::Pl::Tn5 kan-r lacZ. The level of fl-galactosidase in this strain was 807 units. In the next step phage P1 was grown on JHl10 and lysate was used to transduce the wild-type E. coli KL16 selecting kanamycin-resistant colonies on McConkey's agar plates. Colonies showing light blue colour were

294

tested for PUVA. One isolate (JH111) was found to have become PUVA sensitive. It is therefore puoR + puvA::Pl::Tn5 kan-r laeZ, i.e. the expression of lacZ is now under the regulatory control of purR +. 83 units of /3-galactosidase activity were measured in this strain in the absence of any inducers. This construct was subsequently employed in the analysis of gene expression. For this, J H l l l was exposed to MC (5 /~g/ml for varying periods or for 5 h with varying concentrations). The cultures were subsequently analysed for /3-galactosidase activities. Results (Figs. 5 and 6) showed that MC can induce /3galactosidase indicating that the PUV operon is inducible by this agent. Attempts to induce /3-galactosidase by PUVA (20 ~tg/ml psoralen + 200 J / m z NUV) in JH111 showed no induction (data not presented). A plausible explanation for this result is that the cultures could be exposed to PUVA for a short

time only and this exposure is not sufficient to give distinct levels of induction.

Sensitioity of DNA-repair-deficient mutants of E. coli for PUVA To determine which D N A repair genes may be involved in the repair of PUVA induced DNA damage, a number of well identified DNA-repairdeficient mutants of E. coli were tested for sensitivity to PUVA. The mutant strains for recBC, reeF, recN, uorA, recA, recJ, recBC sbcB, and reeBC sbcB recF were exposed to PUVA and their percent survival at various doses determined. It was found that the recA, recBC, recN, recF, uorA and a triple mutant recBC, sbeB reeF were all more sensitive to PUVA than the wild-type KL16. In contrast recJ and recBC sbcB mutants showed wild-type levels of sensitivity (Fig. 7). The data confirms some of the early observations (Sinden and Cole, 1978) that genes responsible for genetic

100-

10-

1-

0.1"

_ lO-2m

.~, 1 0 3-

lo-4 el

10 -5 .

1 0- 6 .

lo-r

12bo jm_2 doo

24bo

3cko

Fig. 4. Bacterial kill curve of F ' , F - and a transconjugant in the presence of PUVA.

295 350

r e c N - derivatives of SA270 are very sensitive to PUVA (Ahmad and Holland, 1985; our unpubfished data) suggesting that resistance in SA270 is not due to a reduction in the ability of the mutant cell to transport psoralen molecules across the cell envelope. Moreover, the enhanced resistance to the structurally unrelated crosslinking agents like NM and MC argues against a permeability change. Alternatively, we might consider that the damaging elements cannot bind to DNA of the mutant bacterium with as high an efficiency as with the DNA of the wild-type parent strain. Finally, the mutant may indeed have an increased efficiency to repair the PUVA damage. From the data presented, we postulate that the enhanced DNA repair is the reason for enhanced resistance towards PUVA. This is supported by the observation that

--~ 300'

E t13 1D

e"

•~

,

200

0

I

¢-

105 -

100 0

4

8

90-

10

gg/ml mitomycin

Fig. 5. Induction of fl-galactosidase controlled by PUV regulatory system in J H l l l in response to different amounts of MC. Culture conditions, MC treatments and units of fl-galactosidase are as described in Materials and Methods.

m ¢u 0~

o70i on

recombination (recF, recBC, etc.) are involved in PUVA-induced DNA repair. In this study we note that mutation in recN also confers sensitivity for PUVA implying a role of this gene in the repair. Also it is interesting to note that the recJ mutant of E. coli is not sensitive to PUVA.

C

50

Discussion

The mutant strain of E. coli, SA270, may have become hyper-resistant to PUVA due to the loss of the ability of the cell to take up psoralen molecules across the cell envelope. From our resuits of uptake experiments with [3H]psoralen we could detect no difference in the uptake of the drug between the mutant and the wild-type strains (unpublished data). Furthermore, recA- and

30

I

I I 2 Time, hours

I 4

I 5

Fig 6. Induction of fl-galactosidase controlled by PUV regulatory system in JH111 in response to different periods of treatment with MC (5 #g/ml). Culture conditions, MC treatments and units of fl-galactosidase are as described in Materials and Methods.

296

100,

• • • o

10.

•

• D o v

0.1

SA270 KL16 recJ recBCsbcB recBCsbcBrecF recF recBC recN uvrA recA

:l m O

600

12(K) j m _ 2

18()0

24()0

30()0

Fig. 7. Bacterialkill curve of a number of repair-deficientmutants of E. coli in the presenceof PUVA.

of the two types of damage, monoadducts (MA) and crosslinks (CL), the mutant bacteria are only able to repair the latter more effectively than its wild-type parent strain. A second question may now be asked, by which mechanism (or pathway) is this repair achieved? Studies have shown that the repair of both types of psoralen induced lesions (MA and CL) involve the u v r A B C excision-repair system, recA protein, D N A polymerase-I, helicase II, and D N A ligase (Cole, 1973; Cole et al., 1976; Van Houten et al., 1986). Based on their studies a model for psoralen crosslink repair has been proposed (Cole, 1973; Van Houten et al., 1986). According to this model, the U v r A B C excinuclease makes incisions on both sides of the pyrone end of the crosslinked base on one DNA strand. Subsequently, the 5 ' ~ 3' excinuclease activity of D N A polymerase-I makes a large gap 3' to the crosslinked site. This gap is a site for recA mediated recombination. The invad-

ing strand displaces the crosslinked incision product. Following the recombination step, the proteins of the u v r A B C complex are free to incise the other strand of the crosslinks. The dual action of D N A polymerase and helicase II leads to the release of an l l - m e r or 12-mer crosslink concomitantly with repair synthesis and the release of the Uur subunits. The repair patch is sealed by the action of D N A ligase. The repair model proposed by Van Houten et al. (1986) suggests that the furan as well as the pyrone ends of the CL are being incised by the u v r A B C excinuclease. Subsequent in vitro studies have shown that this enzyme has little or no incision activity at the pyrone end of the damage (Van Houten et al., 1986). In addition, Jones and Yeung (1990) have shown that the u v r A B C excinuclease activity is selective on incising the psoralen adduct from D N A and is determined by the G C content in the region. From these results it

297 is clear that the uorABC excinuclease m a y n o t b e the sole e n z y m e r e s p o n s i b l e for excising the p s o r a l e n crosslinks. Indeed, Z h e n et al. (1986) have p r o p o s e d a novel glycosylase activity for this repair. F r o m o u r own p r e l i m i n a r y studies on its activity in cell extracts ( u n p u b h s h e d d a t a ) we suggest that the 55-kdal p r o t e i n might be a nuclease specific for the r e c o g n i t i o n a n d incision of D N A at or n e a r the C L s p r o d u c e d b y p s o r a l e n ( a n d p o s s i b l y b y M C a n d n i t r o g e n mustard). G e n e t i c analysis of 55-kdal p r o t e i n has identified a r e g u l a t o r y gene ( p u r R ) a n d a p u t a t i v e structural gene ( p u v A ) l o c a t e d n e a r to each o t h e r at 57.2 m i n on the c h r o m o s o m e m a p of E. coll. Based on the i n d u c t i o n a n d c o m p l e m e n t a t i o n d a t a a m o d e l for the r e g u l a t i o n of the synthesis of 55-kdal p r o t e i n is p r o p o s e d . I n w i l d - t y p e E. coli the synthesis of the 55-kdal p r o t e i n is i n d u c i b l e a n d that the b a s a l level synthesis of this p r o t e i n limits the ability of the cell to incise a n d subseq u e n t l y r e p a i r the C L f r o m the D N A . T h e p u r R gene encodes a r e g u l a t o r y represser p r o t e i n which b i n d s to a p u t a t i v e o p e r a t o r site ( p u v O ) of the P U V operon. Basal level expression of the o p e r o n ensues. I n the p u r R - strain (SA270) the synthesis of this repressor is e l i m i n a t e d (or r e d u c e d ) a n d hence the 55-kdal p r o t e i n (the puvA p r o d u c t ) is synthesised constitutively a n d cells show e n h a n c e d resistance t o w a r d s P U V A . T h e sensitivity of recN m u t a n t s of E. coli for P U V A implies a role of this gene in the r e p a i r b u t further studies are r e q u i r e d to ascertain the m e c h anism. Acknowledgement

T h e a u t h o r s wish to t h a n k the M e d i c a l Research C o u n c i l of G r e a t Britain for their s u p p o r t o f this research b y a g r a n t No. G8611397CA. A l s o Dr. B. S c a n l o n ' s helpful g u i d a n c e a n d Mr. A d r i an T u r n e r ' s assistance in the project is m u c h acknowledged. A l s o are t h a n k e d Dr. B. Bachm a n n , Dr. C l a r k a n d Dr. L l o y d for p r o v i d i n g their valuable bacterial mutants. References

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