In vitro eukaryotic DNA excision repair assays: An overview

Biochimie (1995) 77, 796-802

© So¢i6t6 fran~;aisede biochimie et biologic mol6culaire/ Elsevier, Paris

In vitro eukaryotic DNA excision repair assays: An overview B Sallesa, P F r i t a, C Provotb, J - P Jaega, P C a l s o u a aLaboratoire de Pharmacologic et Toxicologie Fondamentales CNRS, 205, route de Narbonne. 31077 Toulouse Cedex; b$oci6tE Fran~'aise de Recherches et d'lnvestissements (SFRI), Berganton, 33127 Saint-Jean-d'lllac, France

(Received 6 June 1995; accepted 15 September 1995)

Summary - - Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of thls biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts. excision repair / biochemical assay / cell extracts Introduction

DNA is constantly damaged by environmental or endogenous cellular agents. For example, base resi-

dues are frequently modified by oxygen radicals; adducts of purine residues are produced by polycyclic aromatic hydrocarbons, acetyl-aminofluorene and cisplatin, while psoralen derivatives react with pyrimidine bases; ultraviolet light (UV) induces predominantly cyclobutane pyrimidine dimers and (6-4) photoproducts in irradiated DNA. DNA repair is one of the major cellular systems that control genomic stability by preventing the deleterious consequences of all these DNA lesions. Various approaches have been undertaken in order to determine DNA repair activity either directly by measuring the kinetics of disappearence of DNA lesions or indirectly by determining the survival of in vitro damaged plasmids after cell transfection. The biochemical pathways mostly involve,a, in the repair of DNA damage are base excision repair (BER) and nucleotide excision repair (NER). BER consists of two steps: 1) recognition of a narrow spectrum of DNA lesions by specific glycosylases and sequential Abbreviations: NER, nucleotide excision repair; BER, base excision repair; CDDP, cis-dichlorodiammineplatinum(II); AAF, acetyl-aminofluorene; UV, ultraviolet light (254 nm);

UDS, unscheduled DNA synthesis.

cleavage of the N-glycosidic bond and the sugar-phosphate backbone; a~d 2) repair synthesis on a short patch followed by DNA ligation that restores the strand continuity; the complete reaction has been reconstituted recently in vitro using purified enzymes [171. Conversely, NER can recognize a large number of lesions and is performed by a protein complex. This complex allows: l) recognition, incision on both sides of the lesion, excision of the damaged oligonucleotide; and 2) DN ~, polymerization and iigation. NER plays a major role among the repair processes since it recognizes and removes a wide variety of DNA lesions with a similar biochemical mechanism from bacteria to human (for reviews see [21, 26, 27, 42]). Various lines of research have been developed including the use of yeast mutants and human syndromes deficient in NER activity. In the latter case, xeroderma pigmentosum (XP) cells exhibiting UV sensitivity and belonging to different complementation groups have been widely used; some of them are defective in the recognition step (eg XP-A) or in the incision/excision step of NER (eg XP-G and XP-F). The extent of NER activity has also been analyzed in vivo, for instance by quantifying repair synthesis after the excision step (unscheduled DNA synthesis (UDS) assay) and more recently by using repair enzymes able to recognize and incise at ihe site of the lesion (for review see [2]), that allowed to approach the rate of excision of the DNA lesions. The latter analysis has led to the concept that repair efficiency

797 was higher in transcribed than in non-transcribed genes [7, 21 ]. This effect of biological importance has been described as preferential repair. In addition, the transcribed strand of active genes is also preferentially repaired, probably by a direct coupling mechanism between transcription and NER (for reviews see [ 11, 20, 21,431). Numerous insights in the molecular mechanism of the NER process arose from the use of a biochemical assay in which specific repair reactions were detected by radiolabeled repair patches on plasmid DNA when incubated in the presence of transcriptionally active cell extracts [47, 58]. Despite the use of in vitro conditions for the repair reaction with cell extracts, NER on plasmid DNA sufficiently resembles genomic repair since defective repair has been observed with extracts from repair deficient XP cells belonging to complementation groups from A to G [22, 23, 38, 59]. In this paper, we discuss advantages, drawbacks and modifications of this in vitro NER assay.

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The in vitro NER assay initially developed by Wood et al [58] takes advantage of the incorporation of a radiolabeled deoxynucleotide during the repair synthesis step (fig 1A). Briefly, in order to observe repair replication, supercoiled plasmid DNA damaged generally with UV-C light, AAF or CDDP is incubated with a whole cell-free extract in a reaction mixture that includes the four dNTPs, one of them being [~32p] labeled, and ATP together with an ATP-regeneraring system. An undamaged plasmid DNA with a slightly different size is added in the reaction mixture as a control. During the incubation, some DNA lesions are removed by excision repair (NER but also possibly BER, see below). DNA repair synthesis is determined after recovery of plasmid DNA from the mixture, linearization with a restriction enzyme, agarose gel electrophoresis, autoradiography and scintillation counting of the excised bands [58]. The NER activity can be expressed as specific repair synthesis (incorporation in the damaged plasmid minus background incorporation in the control plasmid) or repair factor (ratio of incorporation in damaged versus control plasmid). When radioactive incorporation was observed in plasmid without linearization, the radioactivity associated to repair events was mainly recovered in the closed circular form which indicates that the reaction proceeded through ligation (fig 1B~. It might be interesting to visualize closed-circular and nickedcircular forms and to quantify the radioactivity in each, in order to determine the extent of completion of the repair reaction.

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Fig l. A. Scheme for the repair synthesis assay on damaged plasmid DNA by cell-free extracts (from [58]). Small circles on plasmids represent DNA lesions. B. Kinetics of in vitro repair synthesis reactions on damaged plasmids. 200 l.tg of HeLa cell protein extracts were incubated with 300 ng of damaged pBS plasmid as described [58] except that the untreated control plasmid was omitted. The reactions were stopped at different times and the reaction products were analyzed without (-) or after (+) linearization by EcoRV, by electrophoresis on agarose gel (upper panel) followed by autoradiography of the dried gel (lower panel). Although incorporation of [o~-32P]dAMP was present in open-circular plasmids (OC), the radioactivity primarily accumulated in closed-circular forms (CC) indicating the completion of the reaction. The extent and the specificity of repair synthesis are mostly dependent on the following points: quality of

798 cel!-free extracts and plasmid DNA, DNA lesions and reaction parameters. Extract

Preparation of protein extract might cause variation in repair synthesis activity for instance when nondamage-specific nucl,mses are present. Howe',er, the main fluctuation relied on the cellular origin, numan cell lines exhibiting a higher activity than rodent ones [6]. Cells should be collected during the exponential phase of growth, otherwise the repair ratio decreased due to unspecific nucleases (our unpublished results). Extracts from various tissues could be prepared, but the repair activity was low due to contamination with unspecific nucleases ([32] and our unpublished results). When yeast extracts were assayed, they were first characterized as only proficient for BER in vitro [31, 48, 56]. However, they were shown soon after to perform NER when supplemented with the rate-limiting Rad2 protein [57]. Extracts from tobacco leaves (Nicotiana tabacum) are proficient in the excision repair reaction (our unpublished results). Plasmid DNA

Plasmid DNA should be highly purified in order to remove any breaks from which a DNA polymerization reaction might occur. On the other hand, during the purification procedure (one cesium gradient, two sucrose gradients), DNA fragments from bacterial chromosome that inhibited the reaction must be removed [4]. In the case of damaged plasmid DNA, the purity should be identical to the control, otherwise the observed repair synthesis should not only be due to excision of DNA lesions. Therefore, it is of interest to purify the damaged plasmid DNA on sucrose gradients after the damaging treatment in order to use supercoiled plasmid DNA. Some authors reported that NER 'removed DNA adducts from linear and covalently closed circular DNAs with about the same efficiency' [29]; however, a close inspection of their results showed a significant inhibition of NER with linear plasmids, as described by others ([25] and our unpublished results). Since performing repair on synthetic oligonucleotides is known to alter lesion recognition by modifying the affinity for DNA adducts of repair proteins such as UvrABC [37], we believe that the results of repair experiments with human excinuclease on oligonucleotides should be considered cautiously, especially when the repair efficiency of different classes of lesions is assessed [30]. DNA lesions

UV-C irradiation that induced the formation of pyrimidine dimers was currently used. However, this treat-

ment induced the formation of minor lesions such as strand breaks, thymine glycols and other base modifications from which a label incorporation might occur. Treatment of UV-damaged plasmid DNA with endonuclease III from E coil, which removed thymine glycol, decreased the radiolabeled incorporation [59]. Among various damaging treatments, AAF appeared to induce DNA modifications purely repaired by NER [55]. Most of the damaging treatments induced the formation of a specLrum of adducts that might be differently recognized in the repair reaction. For instance, minor CDDP/DNA adducts were mainly responsible for the repair incorporation [8]. To approach this question, substrates modified with a unique lesion of chemically defined structure were constructed. The use of such substrates showed that 1,2d(GpG), a major CDDP adduct, was not repaired [52] contrarily to 1,3(dGpG) [35]. Cyclobutane thymine-thymine dimer was not repaired while (6--4) photoproduct was [53]. However, when four cyclobutane dimers were present on plasmid DNA, the excision reaction could occur but with an unknown amplitude since the methodology was different from the repair synthesis assay [28]. From the results with UV-treated plasmid, it could be suggested that BER could be performed with extracts of the same type as those used for NER assays. This hypothesis was confirmed with DNA containing uracil [16], apuric/apyrimidic sites [18] and alkylated lesions [40]. These results emphasize the need of extracts from repair mutant cells as control and/or pure NER substrates (AAF-treated DNA) in order to firmly establish the nature (BER or NER) of the repair mechanism responsible for the repair synthesis monitored in vitro. Reaction parameters

The optimal value of various parameters for the in vitro NER reaction has been defined as follows [58]: temperature, 30°C; incubation time, 3 h; KCI concentration, 70 mM with at least !00 [.tg of HeLa cells protein extract per reaction. From various data, it appeared that there might be a threshold in KCI concentration that reduces to a minimum a side damage-dependent nuclease activity present in the extracts that could hide repair synthesis by NER [6~ 15, 58]. The range of K + concentration used wi*.h specific cell-free extracts can be increased when glutamate is used instead of chloride ion [10]. Consequently, when new cell lines are tested, it is necessary to determine the range of salt concentration which gives the best discrimination in repair synthesis between repair proficient and deficient cell extracts. Studies using cell extracts in vitro have proved to be very informative. The incision step appeared to be slow and rate-limiting while gap-filling and ligation

799 proceeded very rapidly ,r~1",7,, moreover, the repair complex might not be processive [54]. The fragment excised by human cell-free extracts ranged in length from 27 to 32 nucleotides [28] and has 5'-P and 3'-OH termini corresponding to enzymatic hydrolysis of mainly the 22-24th and the 5th phosphodiester bonds, 5' and 3' to the lesion respectively [50]. Human single° stranded DNA binding protein (HSSB/RPA) has been involved in carrying out or stabilizing incisions in

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damaged DNA in vitro [ 14, 15, 24] and the proliferating cell nuclear antigen (PCNA)-dependent DNA pol~5/e was shown to be required in vitro for the repair synthesis step [34, 44, 60]. In addition, this system has been used in vitro for purification of protein factors complementing the defect of XP-A and XP-C cell extracts [39, 45], for the identification of the repair defect in XP-G cell extracts [35, 36, 38] and for the characterization of potential repair complexes [5, 55]. More recently, the whole eucaryotic NER reaction has been reproduced in vitro with purified protein components [ 1, 46]. Despite its usefulness, some drawbacks are raised by this assay. From a technical point of view, it is a time-consuming reaction that requires a huge amount of cells and the use of radioactive compounds. Although preferential repair plays a major role in vivo, in vitro NER mimicks more closely global repair of the genome rather than transcriptionally coupled repair. In vitro NER does not reproduce the probable interactions of the repair proteins with chromatin. The repair signal in vitro requires proficient DNA replication in cell extracts. At last, the efficiency of the repair reaction with cell-free extracts has been estimated < 10% with any kind of DNA lesions and is even lower in the reconstituted reaction with purified proteins [1], which implies a role for many unknown accessory factors in vivo.

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Fig 2. Scheme of the 3-D assay procedure. The 3-D assay procedure was the following: 1) plasmid DNA adsorption (1 lxg/ml, 50 l.tl) in sensitized wells; 2) DNA damaging treatment (small circles on plasmid represent DNA lesions); 3) incubation with protein extract and performed as follows: standard 50 l.tl reaction mixture contained 150 lxg extract protein and 70 mM KCI in reaction buffer containing 40 mM Hepes-KOH (pH 7.6), 5 mM MgCI2, 0.5 mM DTT, 10 mM phosphocreatine, 2.5 lxg of creatine phosphokinase, EGTA 2mM, 18 ~g bovine serum albumin, 0.4 I.tM each dGTP, dCTP and dATP and 0.4 ~tM DIG-1 l dUTP. After a 3-h incubation at 30°C, the wells were washed three times with PBS plus 0.1% Tween-20; 4) recognition of digoxygenylated-dUMP by anti-DIG antibody coupled with alkaline phosphatase: the labeled DNA was incubated during 30 min with an anti-digoxygenin conjugated with alkaline phosphatase diluted 1/10000 in PBS plus 0.025% acetylated BSA and 0.1% Nonidet P40. The wells were washed again three times with PBS plus 0.1% Tween-20; and 5) quantification of the light emitted due to dephosphorylation of 'LumiPhos 530' (Lumigen) following 15 min of incubation. The emitted light was measured with a luminometer (Luminoskan, Labsystems) and expressed in relative light unit (RLU). To account for inherent variations due to such an ELISA-Iike assay as well as to subtle differences in the quality of some component of the test that might occur with time, repair activity was expressed as the ratio of RLU in treated versus untreated plasmid DNA.

800 DNA repair synthesis assay with chemiluminescent detection The repair synthesis signal might be used to detect the presence of genotoxic compounds in an in vitro reaction, To take into account all the requirements for a biochemical genotoxicity assay with cell extracts, we have developed a solid-phase instead of a fluid-phase repair assay [40, 41]. The plasmid DNA was adsorbed on a plastic microtiter plate containing 96 wells and then damaged. The 3D assay (damaged D_.NA d._etection assay) procedure was the following (fig2): 1) plasmid DNA adsorption in sensitized wells; 2) DNA damaging treatment; 3) incubation with protein extracts that allowed repair synthesis to occur in the presence of modified nucleotide, for instance digoxigenylated-dUTP (DIG-dUTP); 4) recognition of incorporated DIG-dUMP by an anti-DIG antibody coupled with alkaline phosphatase; and 5) quantification of the light emitted due to dephosphorylation of a chemiluminescent substrate for alkaline phosphatase by the means of a luminometer. The capture of plasmid DNA on sensitized polystyrene wells presents the following advantages: 1) no purification of plasmid DNA is required after the damaging treatment; 2) modified deoxynucleotide might be easily used, allowing chemiluminescent detection; and 3) semi-automation might be performed. Since a wide variety of DNA damages repaired by NER and/or BER are detected and since there is no need of isotopes, the 3-D assay should be broadly used.

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DNA incision assay In order to understand the molecular mechanism of human NER, it is necessary to characterize each step of the reaction independently. However, when NER is performed under the standard in vitro conditions [58], all the steps of the repair pathway are carried out, from the initial lesion recognition and DNA strand incision up to the ligation that restores strand continuity. Therefore the properties of the incision reaction could not be investigated. On the other hand, DNA polymerization-deficient extract but NER-proficient could not be tested since no incorporation would occur. Three different approaches have been used: i) construction of damaged plasmid DNA with oae labeled phosphorus atom near the lesion allowing to detect the excised oligonucleotide [281; fractionation of cell extract to remove the polymerization activity [44]; and 3) in vitro blockade of the repair synthesis step and labeling of the incised intermediates by Klenow polymerase [9]. The last approach was achieved as follows (fig 3A): DNA repair synthesis was inhibited on removal of free

Fig3. A. Scheme for the incision assay on damaged plasmid DNA by cell-free extracts. Small circles on plasmids represent DNA lesions. B. Dose-response for damage-dependent incision activity by NER on psoralentreated DNA. 300 ttg of HeLa cell protein extracts were incubated in the absence of dNTP and with aphidicolin as reported [9] in the presence of 300 ng of pBS plasmid treated to various extents by 8-methoxy-psoralen (8-MOP, gifts of E Sage, lnstitut Curie, Paris) and of 300 ng untreated priM control plasmid. After 3 h at 30°C, plasmids were purified and labeled by Klenow polymerase as described elsewhere [9] in the presence of [tx-3zP]ddATP.After linearization, plasmids were processed by gel electrophoresis on agarose gel (upper panel) followed by autoradiography (lower panel). dNTP together with addition of the DNA polymerase inhibitor aphidicolin to a repair reaction in which cellfree extracts were mixed with undamaged and damaged plasmids. As revealed by the accumulation of open-circular form of the damaged plasmid, a lesion-specific incision activity took place while DNA resynthesis was kept below 5%. In a second step, the

801 incised intermediates were purified and labeled in a polymerization reaction with the Klenow fragment of DNA polI in the presence of radioactive dNTP or ddATE Under the~e conditions, radiolabeling by Klenow polymerase was directly dependent on the number of 3'-OH ends available as primers in incised plasmids and the yield of incision activity was clearly dependent upon the extent of plasmid modification (fig 3B). This method allows the use of any cell-free extract and avoids time-consuming engineering of labeled plasmid DNA [28] or fractionation protocol of the extracts [44]; moreover, labeling at nicked sites in plasmid DNA catalysed by Klenow polymerase allows to assess accurately the incision efficacy in a NER reaction, even when DNA polymerization activity is impaired like in protein extracts from normal lymphocytes [3]. Using this method, we have reported some biochemical properties of damage-specific incision by NER in human cell-free extracts [10] and we have also tested potential incision modulating molecules (our unpublished results).

Conclusion and perspectives Various approaches have been undertaken in order to understand the individual steps of NER in mammalian cells at the molecular level. They take advantage of the availability of several repair mutants in yeast (for review see [51]) and rodent (for review see [13]) and of repair deficient human syndromes such as XP (for review see [12]). The study of mammalian NER has been approached by the cloning of repair genes and the purification of repair proteins (for review see [27]); in parallel, a biochemical analysis of the molecular repair mechanism was developed with a cell-free system that can carry out NER on damaged plasmid DNA using extracts from mammalian cells [47, 58] or from yeast [57]. This assay has been the basis for the biochemical reconstruction of the NER reaction with purified proteins from mammals [ 1] and yeast [ 19]. In order to deal with the whole complexity of the NER mechanism, one needs new assays which should take into account for example the chromatine structure and/or the transcription activity. The in vitro assay has been adapted to chromatin-like DNA instead of naked plasmid DNA but the results have not been improved in term of specificity and extent of repair synthesis [33, 49]. The coupling between repair activity and chromatin modeling is being investigated with Xenopus eggs extract in an in vitro assay (Gaillard e: al, personal communication). In order to evaluate preferential repair, the in vitro NER assay should be modified by using plasmids with eucaryote transcription-regulatory sequences. By combining molecular biological and biochemical approaches with in vitro

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