Robert P.P.Fuchs* and Erling Seeberg1. Institut de Biologie Moleculaire et ... Howard-Flanders et al., 1966; Seeberg et al., 1976). The uvr+ gene products have ...
The EMBO Journal vol.3 no.4 pp.757-760, 1984
pBR322 plasmid DNA modified with 2-acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease
Robert P.P.Fuchs* and Erling Seeberg1 Institut de Biologie Moleculaire et Cellulaire du CNRS, 15, rue Rene Descartes, 67084 Strasbourg Cedex, France, and 'Norwegian Defence Research Establishment, Division for Toxicology, N-2007 Kjeller, Norway *To whom reprint requests should be sent Communicated by K.Kleppe
Covalently closed circular plasmid DNA was treated with reactive derivatives of 2-acetylaminofluorene: N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco-AAEF) and N-hydroxy-N-2-aminofluorene (N-OH-AF), and tested as substrates for the Escherichia coli uvrABC endonuclease and for transformation frequencies on wild-type, uvrA, recA, uvrArecA and polA mutant strains. The uvrABC endonuclease reacted with all three substrates with high efficiency, implicating this enzyme in the repair of DNA containing all three types of adducts. However, only AAF- and AAIF-DNA showed greatly reduced survival on uvrA mutants (five adducts/lethal hit) relative to wild-type (20 adducts/lethal hit). AF-DNA survived equally well on uvrA mutant and wild-type cells, and at a much higher level of modification (60 adducts/lethal hit). A mutation in recA had only a minor effect on the survival of either DNA. The polA mutation reduced the survival of the AAF-treated DNA to the same extent as the uvrA mutation (five adducts/lethal hit). Also AF-DNA showed reduced survival on polA mutant cells versus wild-type. However, many more adducts (20/lethal hit) were tolerated than for AAF-DNA, indicating that AF lesions in the template do not efficiently block replication of DNA. Key words: acetylaminofluorene/aminofluorene/DNA excision repair/plasmid survival three
Introduction In Escherichia coli, repair of DNA damage caused by u.v. and adduct-forming mutagens can be divided into two main pathways, recA +- and uvr+-dependent repair. recA +dependent repair is inducible and includes both recombinational repair and the error-prone SOS repair responses (for a recent review, see Hall and Mount 1981). uvr+-dependent repair is controlled by the products of the uvrA +, uvrB + and uvrC+ genes which together recognize and initiate excision repair of damaged nucleotides (van de Putte et al., 1965; Howard-Flanders et al., 1966; Seeberg et al., 1976). The uvr+ gene products have now been purified and shown to reconstitute a repair endonuclease (uvrABC endonuclease) active on u.v.-irradiated and psoralen plus light-treated DNA (Seeberg, 1978, 1981; Sancar and Rupp, 1983; Seeberg and
Steinum, 1983). 2-Acetylaminofluorene (AAF) is a strong rat liver carcinogen (Miller et al., 1961) and frameshift mutagen (Ames et al., 1973; Santella et al., 1979; Landolf and Heidelberger, 1979) which after metabolic activation forms a major guanine adduct at position C-8 (Kriek, 1965; Kriek et al., 1967; © IRL Press Limited, Oxford, England.
Lefevre et al., 1978). In the experiments described below we have treated plasmid pBR322 DNA with three directly reacting derivatives of AAF, and tested the modified DNA as substrates for the uvrABC endonuclease, and for biological activity when transformed into various E. coli hosts with different repair capabilities. The reactive derivatives used were N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF), its 7-iodo derivative (N-Aco-AAIF) and N-hydroxy-N-2-aminofluorene (N-OH-AF). The enzyme data showed that the uvrABC endonuclease acted upon all three different adducts with approximately the same efficiency. The transformation data showed that recA + was not essential for survival of either modified plasmid DNA, whereas the uvr+ system was required for repair of AAF and AAIF-DNA, but not of AFDNA. Results Strand cleavage of AAF-modified DNA by the uvrABC endonuclease Incubation of AAF-DNA with the uvrABC endonuclease produced on average one strand cleavage per 1.8 AAF adducts for the enzyme concentrations used (Figure 1). No break formation was observed when the DNA was incubated with enzyme in the absence of ATP or with the separated uvrA + or uvrB+ /uvrC+ components of the enzyme. These controls ensured that the cleavage observed was caused by the uvrABC enzyme and not by any contaminating proteins in the partially purified preparations. Incubation of AAIFDNA and AF-DNA with the uvrABC endonuclease gave results similar to those obtained for AAF-DNA (Figure 2).
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Fig. 1. Cleavage of AAF-DNA by the uvrABC endonuclease. Results are plotted as a function of remaining covalently closed DNA relative to that obtained with non-damaged DNA treated with the enzyme (6007o covalently closed DNA). Breaks per molecule was calculated assuming a poisson distribution of breaks in the plasmid DNA preparation. 0: AAF-modified plasmid DNA, treated with uvrABC enzyme in the presence of ATP. 0: AAF-modified plasmid DNA, treated with uvrABC enzyme, no ATP. A: AAF-modified plasmid DNA, treated with uvrA + product in the presence of ATP. 0: AAF-modified plasmid DNA, treated with uvrB+ and uvrC+ products in the presence of ATP.
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R.P.P.Fuchs and E.Seeberg
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Fig. 2. Cleavage of AAIF-DNA and AF-DNA by the uvrABC endonuclease. Results are plotted and breaks per molecule calculated as described in the legend to Figure 1. A,A: AAIF-modified plasmid DNA, treated with uvrABC enzyme in the presence (open) and absence of ATP (closed symbols). 0, 0: AF-modified plasmid DNA, as above. 10050
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Fig. 3. Survival of plasmid DNA (pBR322) damaged with the reactive fluorene derivatives transformed into repair-proficient and repair-deficient cells of E. coli K-12. A: AAF-DNA, B: AAIF-DNA, C: AF-DNA, 0: AB1 157 (wild-type); 0: AB1886 (uvrA), *: AB2463 (recA); 0: AB2480 (uvrA recA).
On average, one break was introduced for every 1.8 and 1.5 adducts, respectively, for AAIF- and AF-treated DNA. Transforming efficiency of AAF-modified DNA on E. coli hosts with different repair capabilities Transforming u.v.-irradiated plasmid DNA has lower survival on uvr- than on uvr+ hosts because of the defect of the uvr cells in repairing the incoming DNA (Roberts and Strike, 1981). Since the uvrABC endonuclease, as judged from Figures 1 and 2, seemed to be involved in repair of AAFmodified DNA, we expected by analogy that such DNA would also have a lower transformation efficiency on uvrmutants. This was the case for AAF-DNA and AAIF-DNA, but not for AF-DNA. When transformed into uvrA cells the number of adducts per molecule at 37/o survival was 5, 7 and 60, respectively, for AAF, AAIF and AF-DNA (Figure 3). The corresponding values for uvr+ cells were 20, 25 and 70, respectively. It thus appears that uvr+ -dependent excision repair is essential for repair of AAF- and AAIF-DNA but not for AF-DNA. Furthermore, even in uvr+ cells there is a greater tolerance for AF-adducts than for AAF- and AAIFadducts. A mutation in the recA gene has only a marginal effect on the transformation efficiency of either DNA, indicating that recA + function is of only minor importance in the repair of transforming plasmid DNA. The same conclusion was drawn by Roberts and Strike (1981) based on their 758
20
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40 60 0 40 60 20 ADDUCTS PER DNA MOLECULE
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Fig. 4. Survival of plasmid DNA (pTS0200) modified with N-Aco-AAF (eft panel) and N-OH-AF (right panel) transformed into DNA polymerase I proficient and deficient strains of E. coli. 0, O; P3478 (polA ); *,E W 3110 (polA +).
studies with u.v. DNA. The transformation response in uvr-hosts suggested that either unrepaired AF-adducts do not block DNA replication, or alternatively that a pathway other than uvrABC exists for the repair of such adducts. To address this question we did experiments similar to those presented above using polA mutant cells as host. The polA gene product, DNA polymerase I, is presumably involved in all types of repair synthesis regardless of the enzyme which initiates excision of the damaged nucleotide residue. Because pBR322 DNA will not replicate in po/A-mutated cells, we used a recombinant plasmid (pTS0200), which carries the E. coli origin of replication in addition to the ColEl replicon (Oka et al., 1980). This plasmid does not require the E. coli polA + gene for its replication. Survival of AAF-DNA is reduced to the same extent by polA as by uvrA consistent with the idea that uvrA and polA mutations represent two different defects in the same repair pathway (Figure 4). For AF-DNA the 37% survival in polA cells corresponds to 20 adducts/molecule relative to 70 adducts/molecule in wild-type cells, which suggests that AF-adducts may be repaired by an excision repair which is uvr +-independent, but requires DNA polymerase I for resynthesis. However, more significant is the finding that many more AF- than AAF-adducts are tolerated even in polA mutant cells, indicating that AF-adducts do not efficiently block replication. Discussion Our previous studies have shown that the uvrABC endonuclease recognizes pyrimidine dimers and psoralenpyrimidine adducts in DNA (Seeberg, 1981). The results presented above show that the uvrABC endonuclease also reacts at purine adducts and thus has a broad substrate specificity towards bulky types of lesions in DNA, irrespective of the base affected and the position at which it is modified. Other recent experiments have shown that the uvrABC endonuclease also reacts with DNA modified with benzo[a]pyrene diolepoxide, which forms a major adduct to guanine at position N-2 (Seeberg et al., 1983), and with DNA damaged by cis-dichloramine platinum (Sancar and Rupp, 1983). AAF binding to guanine has been shown to give rise to a local denaturation extending over several base pairs (Fuchs and Daune, 1972, 1974; Fuchs, 1975; Fuchs et al., 1976). This denaturation is probably due to the insertion of the fluorene ring between the two neighbouring base pairs with concomi-
AAF-modified DNA as a substrate for uvrABC endonuclease
tant flipping out of the modified guanine residue (insertiondenaturation model, Fuchs et al., 1976). Both AAIF and AF binding induce much less distortion of the double helix and an outside binding model for both of these adducts has been postulated (Fuchs and Daune, 1973; Fuchs et al., 1976; Evans et al., 1980; Leng et al., 1980; Daune et al., 1981). In this model the fluorene ring is located in the DNA large groove and the modified guanine residue is stacked between the neighbouring base pairs. The observation that the uvrABC endonuclease acts at all three types of adducts with approximately the same efficiency suggests that the uvrABC enzyme does not specifically recognize a particular type of conformational change in DNA, but rather acts at any base damage causing significant distortion of the helix structure. Plasmids damaged by AAF, AAIF or u.v. (Schmid et al., 1982; Roberts and Strike, 1981) all survive -20 lesionsAethal hit in a wild-type host, indicating saturation of the excision repair capacity, since only 3-5 adducts are tolerated in excision-defective (i.e., uvrA) cells. In contrast, irrespective of the repair capacity of the host (wild-type, uvrA, recA, polA, uvrArecA), as many as 20-70 AF-adducts are tolerated per lethal hit, suggesting that this particular adduct is not an efficient block of replication in vivo. Both experimental data (Evans et al., 1980; Leng et al., 1980) as well as theoretical calculations (Broyde and Hingerty, 1983) indicate that AF-adducts in contrast to AAF-adducts do not induce the anti-syn conformational change. This means that the affected guanine residue remains in its normal position in the template structure, which could explain why AF-adducts do not appear to block replication. Although AF-DNA and AAIF-DNA were found to have a similar conformation in double-stranded DNA, it is likely that their respective conformations are quite different in single-stranded DNA. Indeed, AAIF is prevented from insertion into double-stranded DNA due to sterical hindrance of the iodine atom, however, in single-stranded DNA, i.e., at the replication fork, the AAIF residue may adopt a syn conformation similar to that of the AAF-adduct and therefore block replication. The anti conformation of AF-DNA can be stabilized through a hydrogen bond between the amino hydrogen of AF and the 5-0 atom of deoxyribose (Evans et al., 1980) both in double- and single-stranded DNA. Another observation which supports the notion that AFadducts do not block replication is that treatment of cells with N-OH-AF, in contrast to N-Aco-AAF and u.v.-light, does not induce high levels of recA protein (Salles et al., 1983). The single-stranded gaps that are formed in replicating DNA in u.v.-irradiated cells, and presumably also in N-Aco-AAF treated cells, are thought to act as signals for recA induction. The observation that AF does not induce recA suggests that such gaps are not being formed in N-OH-AF-treated cells. After the experimental part of this work had been completed, Tang et al. (1982) reported measurements of transfection of phage OX174 DNA treated with either N-Aco-AAF or N-OH-AF. They showed that AAF-DNA transfected uvrA cells with much lower frequency than uvr + cells while AFDNA showed higher and equal survival on both uvrA and uvr+, consistent with the transformation data presented here. These authors further reported that AF-DNA had lower survival on uvrC- than on uvrA- or uvrB-mutated cells, and postulated that uvrC+ , but not uvrA + and/or uvrB+, was involved in AF-DNA repair. Our results indicate that the uvrABC endonuclease is involved in repair of AF-adducts
even though it is not essential for survival. In view of the results of Tang et al. (1982), we tested survival of AF-treated plasmid DNA on uvrC hosts and confirmed that survival was lower on uvrC than on uvrA hosts. However, we have not detected any repair endonuclease of glycosylase activity of the uvrC protein on AF-DNA in vitro (data not shown). It could be speculated that the uvrC protein has some function in the cells other than being part of the uvrABC endonuclease, and it could even be involved in replication past such lesions in the DNA. Further work is needed to examine this possibility.
Materials and methods Strains Strains used for the transformations were ABI157 (wild-type), AB1886 (uvrA), AB2463 (recA) AB2480 (uvrArecA) and P3478 (potA 1). All were obtained from Paul Howard-Flanders. Chemicals
N-Acetoxy-N-2-acetylaminofluorene [3H-ring] (N-Aco-AAF [3H-ring]), N-acetoxy-N-2-acetylamino-7-iodofluorene [3H-ring] (N-Aco-AAIF [3Hring]) and N-hydroxy-aminofluorene [3H-ring] (N-OH-AF [3H-ring]) were synthesised according to published procedures starting from a 2-nitrofluorene [3H-ring] sample (sp. act. 200 mC/mmol) (Patrick et al., 1974; Lefevre et al., 1978). DNA Plasmid DNA was purified from 1 liter cultures of E. coli AB1 157 strain transformed with pBR322 (Bolivar et al., 1977) or pTS0200 (Oka et al., 1980) by a NaCl/SDS lysis procedure (Seeberg, 1978) followed by a CsCl/ethidium bromide centrifugation (Katz et al., 1973). 14C-Labeled plasmid DNA (sp. act. 2.2 x 104 d.p.m.4tg) was similarly obtained from a CR 34 (thy-) E. coli strain transformed with the pBR322 and grown in presence of 14C-labeled thymidine. Carcinogen modification ofplasmid DNA N-Aco-AAF [3H-ring] and N-Aco-AAIF [3H-ring] reaction with supercoiled plasmid DNA was performed in sodium citrate buffer 2 x 10-3 M, pH 7.0 containing 5% ethanol. DNA concentration was 50 yg/ml, and carcinogen 42.5 gM. The carcinogens were allowed to react for various periods of time and unbound fluorene derivatives were removed by four successive ethanol precipitations. N-OH-AF [3H-ring] was reacted with plasmid DNA under the same conditions except that pH was 5.0. Unbound N-OH-AF was removed by one phenol-sevag extraction, followed by two sevag extractions and three ethanol precipitations. The quantification of the number of adducts per molecule was made as previously described (DeMurcia et al., 1979). The number of adducts per plasmid molecule when the 14C-labeled plasmid DNA was used, was calculated from the data obtained with a cold plasmid reacted under the same conditions. E. coli transformation E. coli cells were made competent for transformation by treatment with CaCl2 (Cohen et al., 1972). Transformants were selected on LB plates containing ampicillin (50 g/ml). Treatment with E. coli uvrABC endonuclease The uvrA +, uvrB+ and uvrC+ gene products were extracted and partially purified as previously described (Seeberg, 1978) except that strain JC6720 (recB recC endA) was used as enzyme source. The cells were u.v.-irradiated (60 J/m2) in mid-log phase and incubated for I h prior to protein extraction to increase levels of uvr gene products in the cells by SOS induction (Kenyon and Walker, 1980). Conditions for the endonuclease treatment were as previously described (Seeberg, 1978) and the plasmid DNA was analyzed for strand breakage by sedimentation in alkaline sucrose as published (Seeberg, 1981).
Acknowledgements We are grateful to Anne-Lill Steinum for carrying out periments reported here.
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Received on 8 December 1983
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