Formation, detection and repair of AP sites

Mutation Research, 181 (1987) 45-56 Elsevier

45

MTR 04415

Formation, detection and repair of AP sites Myriam Talpaert-Borl6 Laboratory of Biochemistry, Biology Group, Ispra, Directorate General XII/F, Commission of the European Communities, Joint Research Centre, 1-21020 Ispra (Va) (Italy) (Received 5 January 1987) (Revision received 27 March 1987) (Accepted 7 April 1987)

Keywords: Apurinic/apyrimidinic sites; AP sites; DNA; Methoxyamine.

Summary The paper is an outline review of the main aspects concerning the formation and repair of AP (apurinic/apyrimidinic) sites in DNA as well as some of the chemical properties allowing their quantitative determination. A new method for the measurement of AP sites based on their reaction with [14C]methoxyamine is described. It has been applied to the measurement of AP sites produced in DNA either by physical (-/-rays) or chemical (methyl methanesulphonate, osmium tetroxide) agents. The method has also been used to quantify the excision of abnormal bases from DNA under the action of specific DNA glycosylases and to prevent the chemical or enzymatic degradation of DNA containing AP sites. The paper contains data about the purification and characterization of uracil-DNA glycosylase and AP endodeoxyribonuclease from carrot cells, two enzymes involved in the first steps of base excision repair through AP site intermediates. The biological effects of unrepaired AP sites are also discussed.

(I) Introduction The most important DNA repair pathway proceeds via the excision of an altered residue or group (Fig. 1). The formerly proposed DNA excision scheme, nucleotide excision repair, involves the recognition of a damaged nucleotide by an incision endonuclease, the excision of a DNA fragment comprising the damaged region, and then the reconstruction of the missing part by DNA polymerase and DNA ligase. The best example is

Correspondence: Dr. Myriam Talpaert-Borl6, Laboratory of Biochemistry, Biology Group, 1-21020 Ispra (Va) (Italy).

the pyrimidine dimer excision by the UVR incision system of Escherichia coli. Another pathway of excision repair has been proposed after the discovery of DNA glycosylases, a new class of enzymes, which recognize a specific abnormal base and remove it from DNA leaving an apurinic/apyrimidinic (AP) site (Lindahl, 1976). This repair pathway, called base excision repair (Duncan et al., 1976), subsequently involves the incision of the phosphoester bond adjacent to the AP site by an AP endonuclease, the excision of the baseless site, and the successive action of DNA polymerase and DNA ligase as in nucleotide excision repair. Although there is a paucity of information concerning enzymes implicated in the DNA repair of

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

46

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plant cells, it is now well established that higher plants are able to repair damages caused by UV, ionizing radiation or chemicals by mechanisms similar to those described in bacterial and mammalian cells (Veleminsky and Gichner, 1978). The knowledge on DNA repair in plant cells has been recently reviewed by Osborne et al. (1984). The identification of an AP endonuclease in tissues of Phaseolus multiflorus (Verly et al., 1973; Thibodeau and Verly, 1976, 1977) and in barley (Veleminsky et al., 1977, 1980; Svachulova et al., 1978) suggested the occurrence of AP sites in plant cells as well. Nuclease for DNA apurinic sites may be involved in the maintenance of DNA in normal cells. The central role of AP sites as intermediate in DNA repair of damage caused by physical and chemical agents has been acknowledged also in plant cells. As a matter of fact, recently a uracil-DNA glycosylase has been isolated from carrot cells (Talpaert-Borl6 and Liuzzi, 1982) and from wheat germ (Blaisdell and Warner, 1983). Here we give an outline review of the main aspects concerning the formation and repair of AP sites, as well as some of the chemical properties allowing their quantitative determination. Some of our research data are also presented. (2) Formation of AP sites

AP sites are very frequent DNA lesions resulting from the cleavage of the glycosyl bond be-

tween deoxyribose and purines or pyrimidines. The base loss may be spontaneous even at normal pH (Lindahl and Nyberg, 1972). It is enhanced by chemicals such as alkylating agents (Lawley and Brooks, 1963), and by physical treatments such as irradiation (Dunlap and Cerutti, 1975; T6oule et al., 1977) which cause some base modifications responsible for the weakening at the glycosyl bond. Furthermore, AP sites may also result from the enzymatic excision of abnormal or altered bases catalysed by specific DNA glycosylases (Lindahl, 1976).

(2.a) Spontaneous depurination / depyrimidination The base-sugar bonds in DNA are susceptible to acid hydrolysis (Tamm et al., 1952). Purines are released under weak acid conditions while pyrimidines are released under conditions of higher acidity. Moreover, purine bases are lost from DNA during incubation at neutral pH and high temperature (Greer and Zamenhof, 1962). Lindahl and Nyberg (1972) have measured a rate constant for DNA depurination of 4 x 10-9/sec at 70°C and pH 7.4 in a Mg2+-containing buffer of physiological ionic strength. The activation energy of the reaction is 130 kJ/mole. From these data it has been estimated that an E. coli cell, growing with a generation time of 40 min at 37°C should lose 0.5 purine/chromosome in each generation and that the depurination of DNA in mammalian cells should amount to 5000-10 000 events/day. The spontaneous depyrimidination of DNA also occurs at neutral pH, but at a 20 times slower rate than depurination (Lindahl and Karlstr6m, 1973).

(2.b) Depurination and depyrimidination by chemical agents Treatment of DNA with alkylating agents (methyl methanesulphonate, dimethyl sulphate, etc.) causes base alkylation, mainly at purine residues (Lawley and Brooks, 1963). The alkylated bases (7-methyladenine, 3-methyladenine, 7-methylguanine, 3-methylguanine, etc.) are positively charged. This results in a weakening of the glycosyl bond and an increase in the release of bases (Strauss and Hill, 1970). Fig. 2 shows the depurination of DNA treated with methyl methanesulphonate (MMS). The number of AP

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Fig. 2. Depurination of DNA by methyl methanesulphonate. Unlabelled or [3H]DNA were treated with concentrations of methyl methanesulphonate (MMS) from 0 to 0.3 M during I h at 37 o C, dialysed against 0.5 M NaC1, 0.015 M sodium citrate, pH 7.0, and heated at 50 ° C for 6 h (Paquette et al., 1972). AP sites were measured either from the [14C]methoxyamine incorporation in acid-insoluble material after a 30-min incubation at 37°C with 5 mM [14C]methoxyamine ( x ) or from the acidsoluble radioactivity from [3H]DNA after a 15-rain incubation at 37°C without (O) or with 0.2 N NaOH (O) as described previously (Talpaert-Bod6 and Liuzzi, 1983; Liuzzi and Talpaert-Borl6, 1985). Unpublished results.

sites, measured either from their reaction with [14C]methoxyamine or from the acid-soluble fraction resulting from the alkaline breakage o f the adjacent phosphoester bond (see Section 3), increases with the alkylating treatment. Treatment of DNA with nitrous acid also leads to base loss. It occurs through the deamination of guanine to xanthine residues which are easily lost from DNA (Schuster, 1960). Oxidizing agents such as osmium tetroxide, potassium permanganate or hydrogen peroxide cause DNA depyrimidination due to the weakening of the glycosyl bond between deoxyribose and modified thymine residues with saturated 5,6 bonds (Iida and Hayatsu, 1971; Demple and Linn, 1982). Fig. 3 demonstrates that depyrimidination of DNA treated with osmium tetroxide (OsO4) increases with the reagent concentration. AP sites can also be introduced in DNA by some antitumour antibiotics such as bleomycin which binds to DNA and causes the breakage of thymine-deoxyribose bonds (Miiller et al., 1972).

(2.c) Depurination and depyrimidination by irradiation AP sites are part of the alkali-labile sites pro-

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Fig. 3. Depyrimidination of DNA by osmium tetroxide. Aliquots of 500/~1 containing 150/tg of unlabelled or [3H]DNA in 0.010 M NaC1, 0.010 M sodium phosphate, pH 7.2, were partially denatured with 1 ml of 0.3 M potassium phosphate, pH 12.4, and treated with 0-5 m g / m l osmium tetroxide (OsO4) for 30 min at room temperature. The samples were neutralized by adding 750 ttl of 1 M potassium phosphate, pH 4.0. OsO4 was removed by 3 extractions with 3 vol. of ether followed by an extensive dialysis against 0.010 M NaCI, 0.010 M sodium phosphate, pH 7.2. AP sites were measured either from the [14C]methoxyamine incorporation in acid-insoluble material after a 30-min incubation at 37°C with 5 mM [lac]methoxyamine ( × ) or from the acid-soluble radioactivity from [3H]DNA after a 15-rain incubation at 37°C without (o) or with 0.2 N NaOH (©) as previously described (Talpaert-Bod6 and Liuzzi, 1983; Liuzzi and Talpaert-Bod~, 1985). Unpublished results.

duced in DNA by ionizing radiations (Ljungquist et al., 1974). The hydroxyl radicals formed during irradiation in aqueous medium can cause the cleavage of the glycosyl bond either by their direct attack on the bond, or, as a consequence of their reaction with the base moieties resulting in the weakening of the glycosyl bond (Dunlap and Cerutti, 1975; T6oule et al., 1977). Fig. 4 shows that y-rays introduce in DNA a dose-dependent number of lesions leading to the formation of deoxyribose aldehyde groups involved in the formation of strand breaks under alkaline conditions, or in the reaction with specific aldehyde reagents such as methoxyamine (see Section 3). These aldehyde groups result either from the release of damaged bases leaving intact doxyriboses (i.e. AP sites), or from the deoxyribose oxidation leading to the release of intact bases and to the formation of one or more aldehyde groups (Von Sonntag et al., 1981). Apyrimidinic sites are also formed in DNA by

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Fig. 4. Formation of aldehyde groups, strand breaks and alkali-labile sites in 3,-irradiated DNA. (A) Aldehyde groups in unlabelled DNA (450 ~g/ml in 0.010 M NaC1, 0.010 M sodium phosphate, pH 7.2) irradiated with increasing doses of 3,-rays in the presence of 02 (I), N 2 without (©) or with 0.3 M cysteamine (×) measured from the [14C]methoxyamine incorporation in acid-insoluble material after a 30-min incubation with 5 mM [14C]methoxyamine as previously described (Talpaert-Borl~ and Liuzzi, 1983). (B) Strand breaks and alkarl-labile sites in [3H]DNA (450 /xg/ml in 0.010 M NaC1, 0.010 M sodium phosphate, pH 7.2) irradiated with increasing doses of "/-rays in the presence of O2 measured from the acid-soluble radioactivity after a 15-min incubation at 37 °C without (e) or with 0.2 N NaOH (©) as previously described (Liuzzi and Talpaert-Borl& 1985). Unpublished results.

irradiation with high doses of ultraviolet rays (Ljungquist et al., 1974). They presumably result from the spontaneous release of oxidized pyrimidine derivatives.

(2.d) Depurination and depyrimidination by specific DNA glycosylases Altered or a b n o r m a l bases in D N A can be removed enzymatically by specific D N A glycosylases (Lindahl, 1976). These enzymes catalyse the cleavage of the glycosyl b o n d between the deoxyribose and the base residues leaving the phosphodiester b a c k b o n e intact. The p r o d u c t s of the glycosylase catalysis are a free base and an A P site. Some 10 specific D N A glycosylases have been r e p o r t e d , i n c l u d i n g those w h i c h r e c o g n i z e deaminated bases such as uracil or hypoxanthine, alkylated purines such as 3-methyladenine, ringopened fragmented purine such as formamido pyrimidine, or pyrimidine fragment such as urea, ring-saturated pyrimidine such as thymine glycol,

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Fig. 5. Depyrimidination of uracil-containing polydeoxyribonucleotide by uracil-DNA glycosylase.The uracil-DNA glycosylase reaction was performed in a mixture containing Tris-HC1, EDTA, and dithiothreitol as in the standard enzyme assay with poly(dA-dT, [3H[dU), (dT:dU=12) at a concentration of 520 #M as total nucleotides. After incubation at 37°C either in the presence of 5 #g protein/ml enzyme preparation (Sepharose-poly(rU) fraction) during the indicated times (A), or during 30 min in the presence of the indicated amounts of protein (B), the enzyme was inactivated by a 5-min heating at 70 o C in the presence of 0.5 M KC1 and the mixture treated with 5 mM [14C]methoxyamine during 30 min at 37°C. The remaining [3H]uracil ([3H]Ura; @) and the [14C]methoxyamine-reacted AP sites ([14C]M-AP site; (3) were determined by measuring the acid-insoluble 3H and 14C radioactivities. From Liuzzi and Talpaert-Borl~ (1985).

or pyrimidine dimers. T h e s e e n z y m e s are ubiquitously distributed and have very similar physical and biochemical properties which have been extensively reviewed by Lindahl (1979, 1982). T h e y have a low molecular weight (between 18 000 and 31 000). They act by simple hydrolytic cleavage of the glycosyl bond, generally on doublestranded D N A configuration, without requirements for cofactors such as divalent cations. In spite of these similarities, these enzymes have a narrow substrate specificity. Fig. 5 shows the release of [3H]uracil from a uracil-containing polydeoxyribonucleotide and the concomitant formation of A P sites by incubation with u r a c i l - D N A glycosylase. The kinetics are a function of time and enzyme concentration. F o r each point there is a good correspondence between the n u m b e r of [14C]methoxyamine-reactive A P sites and [3H]uracil released as deduced from the residual [3H]uracil content in the polydeoxyribonucleotide.

49

(3) Chemical properties of AP sites The deoxyribose residue at an AP site is in equilibrium between the furanose ring and an open form with a free aldehyde group on the C~ and an alcohol function on the C4 (Overend, 1950). The presence of these chemical groups accounts for the great reactivity of AP-DNA. The properties of apurinic and apyrimidinic sites in DNA have been reviewed (Lindahl and Ljungquist, 1975). Here only the properties of AP sites related to their quantitative determination will be mentioned.

(3.a) Cleavage of the phosphoester bond adjacent to an A P site The aldehyde group on C~ promotes a fl elimination reaction in the presence of nucleophilic molecules such as thiols, primary and secondary amines, polyamines (Lindahl and Anderson, 1972), basic tripeptides (Behmoaras et al., 1981a,b; Pierre and Laval, 1981), basic proteins such as lysozyme, cytochrome c, ribonuclease (Thibodeau and Verly, 1977; Pierre and Laval, 1981) or several aldehyde reagents (Livingston, 1964). The C3 phosphate elimination leads to the breakage of the phosphoester bond on the 3' side of the AP site producing a 5'-phosphate end and an a,fl-unsaturated deoxyribose derivative. In the presence of excess fl-eliminating agent, a second phosphate elimination occurs on the 5' side generating a C3 phosphorylated terminus and an unsaturated sugar residue (Grossman and Grafstrom, 1982). (3.b) Addition o) aldehyde reagents on A P sites The aldehyde group on C~ of an AP site can react with nucleophilic reagents of aldehydes containing an - N H 2 group. In the case of compounds with - N H - N H 2 groups, the addition is followed by the elimination of water and by the formation of a Schiff base which promotes the fl elimination reaction leading to the cleavage of the phosphoester bond and the degradation of DNA. Coombs and Livingston (1969) showed that the aldehyde reagents with - O N H 2 groups such as hydroxylamine or methyl hydroxylamine (methoxyamine) react with the deoxyribose aldehyde group without degrading DNA. The stabilization of the apurinic acid by these derivatives is prob-

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apyrimidinic sites (B). The reaction mixtures contained 200 g g / m l of either untreated or alkylated-depurinated DNA (A), 175 g g / m l of poly(dA)E30-poly(dT, dU)230 either treated or untreated with uracil-DNA glycosylase (B). The incubation was at 37°C, pH 7.2, with 1 mM (@) or 5 mM (©) [14C]methoxyamine for the treated polymers and with 5 mM (e) for the untreated ones. At the indicated times, the incorporation of [14C]methoxyamine was measured from the acidinsoluble radioactivity. Inserts of A and B: Number of unreacted sites ( F ) as a function of time. From Talpaert-Borl6 and Liuzzi (1983).

ably due to the formation of a hydrogen bond between the - O H group, which appears on C~, and the oxygen of the reagent preventing the loss of water and the formation of a Schiff base. We have investigated the reaction of apurinic and apyrimidinic sites with methoxyamine in order to define the conditions necessary for their quantitative determination (Talpaert-Borl~ and Liuzzi, 1983). Studies with alkylated-depurinated DNA and with a uracil-containing polydeoxyribonucleotide with apyrimidinic sites show

50

that methoxyamine reacts quickly and exhaustively with both apurinic and apyrimidinic sites (Fig. 6). The reaction is irreversible and the complex cannot be hydrolyzed at pH 7.2. Methoxyamine can be removed from the deoxyribose by the addition of molecules containing aldehyde groups such as acetaldehyde (Liuzzi and Talpaert-Borl~, 1985). Hadi and Goldthwait (1971) have found that hydroxylamine-treated depurinated DNA is not degraded by alkali. Our results show that the addition of methoxyamine on the AP sites prevents the alkaline as well as the enzymatic breakage of the adjacent phosphoester bond (Liuzzi and Talpaert-Borl~, 1985). (4) Measurement of AP sites (4.a) Measurement of AP sites from the determination of strand breaks Most of the methods measuring AP sites are based on the determination of the number of strand breaks resulting from the rupture of the phosphoester bonds adjacent to the AP sites. The most widely used alkaline treatment may be replaced by one using polyamines or specific endonucleases (Brent et al., 1978; Helland et al., 1982). The number of breaks introduced in a heterogeneous population of labelled E. coli DNA can be estimated from the acid-soluble radioactivity (Paquette et al., 1972). Bricteux-Gr6goire et al. (1986) have established the relationship between DNA acid solubility and the frequency of singlestrand breaks near apurinic sites (Fig. 7). Such a method is not very sensitive because the break frequency must be high to have a measurable acid-soluble radioactivity. A more sensitive but also long and tedious method is that using the sedimentation of a homogeneous population of linear T7 phage DNA through neutral sucrose gradient (Crine and Verly, 1976). Filtration through nitrocellulose or agarose gel electrophoresis (Goffin and Verly, 1982; Bradley et al., 1982) allows the determination of the number of strand breaks in supertwisted RF-I DNA of various origins. On the other hand, the number of strand breaks in DNA of uniform length although under various structural forms (supercoiled, par-

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Fig. 7. Single-strand break frequency and acid solubility of methylated-depurinated DNA. 3H-Methylated [32p]DNA in SSC was depurinated by heating at 50 o C. Samples were taken at intervals, incubated at alkaline pH to place a break near each AP site before the solubility (%) of the 3Hmethylated-depurinated [32p]DNA was measured. The number N of single-strand breaks is the average of 2 independent determinations: loss of [3H]methyl groups corrected for the number of AP sites already in the 3H-methylated [32p]DNA; AP sites counted with [14C]methoxyamine. The results from 2 different Expts. (Table I: C)= Expt. I; /, = Expt. II) are distributed along a sigmoidal curve. From Bricteux-Gr6goire et al. (1986).

tially relaxed, relaxed, linear) can be determined after separation of intact and nicked molecules by electrophoresis on alkaline agarose gel (Ciomei et

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Fig. 8. Incorporation of [14C]methoxyamine as a function of the reactive sites in alkylated-depurinated DNA. The reaction mixtures contained various amounts of reactive sites obtained by mixing various proportions of untreated and alkylated-depurinated D N A at the concentration of 115/~g/ml. The reactions were performed under standard conditions: 5 mM [14C]methoxyamine/0.1 M borate buffer (pH 7.2), 30 rain at 37 o C. The incorporation of [14C]methoxyamine in D N A was measured from the acid-insoluble radioactivity. From Talpaert-Borl6 and Liuzzi (1983).

51 al., 1984). These methods are sensitive but are limited to only a very narrow range of break frequencies. All the methods cited above are indirect methods measuring only intact AP sites, i.e. those which are not already associated with strand breaks. Moreover, those using alkaline treatment do not measure specifically the pre-existing AP sites. They also include the alkali-labile sites due to the degradation of altered bases formed during the alkaline treatment as observed for irradiated or alkylated DNA containing a great deal of damage (Duker et al., 1982).

(4.b) Measurement of AP sites from their labelling with [14C] metho xyamine Recently we standardized a method allowing the direct measurement of aldehyde groups in DNA, especially AP sites, either intact or already associated with strand breaks (Talpaert-Borl6 and Liuzzi, 1983). It is based on the reaction of [a4C]methoxyamine with the aldehyde group present on the deoxyribose moiety after a base loss. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37 ° C, pH 7.2), untreated DNA is not reactive and the [a4C]methoxyamine incorporation in DNA is proportional to the number of AP sites (Fig. 8). Since methoxyamine does not cause the degradation of DNA, the reacted AP sites may be counted by a simple determination of the acid-insoluble radioactivity.

(5) Enzymatic repair of AP sites The AP sites, common intermediates during the repair of damage caused by various agents, are quickly repaired in vivo. The two distinct mechanisms by which the repair proceeds have been extensively reviewed (Lindahl, 1979, 1982; Friedberg et al., 1981; Linn, 1982).

(5.a) Degradative pathway via phosphodiester bond cleavage The correction of AP sites in DNA is initiated by specific endonucleases which catalyse the cleavage of the phosphodiester bonds specifically at sites of base loss. The AP endonucleases which are ubiquitously distributed in nature are of two types: those that

cleave DNA at the 3' side of the AP site and those that cleave at the 5' side. In both cases, a 5'-phosphate and a 3'-OH group appear at the cleavage site. Most of the AP endonuclease activity present in cell extracts from either bacterial or mammalian cells is of the second type. It is presently unclear if the deoxyribose-5-phosphate could be excised from the DNA by the concerted action of the two types of AP endonuclease (Gordon and Haseltine, 1981), or by one of the cellular exonucleases acting after DNA cleavage by an AP endonuclease. It is worthwhile to note that a homogeneous AP endonuclease from human placenta has been found to incise DNA at both sides of an AP site, although the 5' side is the preferential one (Grafstrom et al., 1982). After excision of the baseless sites, the repair continues by the successive action of DNA polymerase which fills the gaps and DNA ligase which seals the nicks.

(5.b) Conservative pathway via reinsertion of the missing base Purine residues have been found to be reinserted into un-nicked apurinic sites in DNA by extracts from E. coli (Livneh et al., 1979) and from human fibroblasts (Deutsch and Linn, 1979a,b). Nevertheless, the existence of the enzyme responsible for this activity, called insertase, has been controverted. Kataoka and Sekiguchi (1982) have demonstrated that the repair attributed to an E. coli insertase is carried out through a short-patch excision pathway with replacement of a single nucleotide. Moreover, it has been suggested that in human cells the apparent insertion of guanine could occur through a noncovalent binding to DNA. As a matter of fact, it occurs without a cofactor or energy requirement (Lindahl, 1982).

(6) Biological effects of unrepaired AP sites Although the cells contain active excision repair mechanisms which restore the normal base sequence, some unrepaired AP sites may be encountered by the replicating enzymes. The consequence for the cell is toxicity or lethality by mechanisms not yet completely elucidated. AP sites are known to cause DNA inactivation (Brakier and Verly, 1970) owing to the production

52 of strand breaks (Lindahl and Anderson, 1972) or interstrand DNA crossfinks (Burnotte and Verly, 1972; Goffin and Verly, 1983). Moreover, AP sites in the DNA template constrain RNA polymerase to pause causing a decrease in RNA synthesis (Flamre and Verly, 1985). Evidence about the role of depurination in spontaneous as well as chemically induced mutagenesis has been recently reviewed by Loeb (1985). In vitro experiments have shown that AP sites may be by-passed by D N A polymerase resulting in misincorporation of nucleotides (Shearman and Loeb, 1975; Boiteux and Laval, 1982). The D N A polymerase ability to copy past apurinic sites, misincorporating preferentially deoxyadenosine, is related to the intrinsic accuracy of the enzyme essentially due to its proofreading capacity (Kunkel et al., 1983). Procaryotic DNA polymerase exhibits high fidelity and copies past these sites with difficulty while, in vitro purified eucaryotic DNA polymerases are error-prone and copy past them readily. In vivo experiments have shown that, in E. coli spheroplasts, the error-prone DNA synthesis on depurinated DNA occurs upon induction of the SOS response (Schaaper and Loeb, 1981; Schaaper et al., 1982); the essential role of AP sites has been clearly demonstrated (Schaaper et al., 1983). In eucaryotic cells it is not clearly established whether the mutagenicity of AP sites depends on the induction of an SOS response. Gentil et al. (1984) have shown that AP sites in SV40 DNA are highly mutagenic in animal cells. The mutagenesis seems to occur without induction of an error-prone DNA repair pathway. According to S u e t al. (1985) AP sites i n alkylated singlestranded DNA of parvovirus H1 have a mutagenic potential whose expression in human cells is triggered by the UV irradiation of the cell.

(7) Base excision repair of uracil-containing DNA by enzymes from plant cells There are several pathways for the introduction of uracil residues into DNA. Cytosine spontaneously deaminates to uracil at a slow but significant rate increasing under the action of heat (Shapiro and Klein, 1966; Lindahl and Nyberg, 1974) or chemical agents such as bisulphite or nitrous acid (Shapiro et al., 1973). The result is a

stable U : G base pair that would then lead to a C : G ~ T : A transition mutation after the next round of replication (Shapiro, 1977). Moreover, deoxyuridylate can occasionally be incorporated in newly synthesized DNA instead of deoxythymidylate (Bessmann et al., 1958). In spite of these facts, uracil does not occur as a normal base in most DNA since it is efficiently removed by an active base excision repair mechanism involving the action of uracil-DNA glycosylase and AP endonuclease (Lindahl, 1979, 1982). These enzymes originally found in procaryotes, then in mammalian cells, have been identified also in plant cell extracts. Using extracts from cultured carrot cells we have isolated and purified an uracil-DNA glycosylase and an AP endonuclease (Talpaert-Borl6 and Liuzzi, 1982). The two activities belong to two distinct proteins which can be separated by affinity chromatography on Sepharose-poly(rU) for which the glycosylase has a great affinity (Fig. 9). The two enzymes were found to have properties similar to those of the homologous bacterial and mammalian enzymes.

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Fig. 9. Separation of uracil-DNA glycosylase and AP endonuclease from carrot cells by chromatography on Sepharose poly(rU). 2.5-ml samples were collected. O, absorbance at 280 nm; - - , potassium phosphate linear gradient; O, chromatographic profile of AP endonudease activitydeterminedby measuring the acid-soluble radioactivity released from alkylated-depurinated [14C]DNA after a 15-rain incubation at 37°C with an aliquot from each collected fraction diluted 8-fold in the standard assay; x, chromatographicprofile of uracil-DNA glycosylaseactivity determined by measuring the [3H]uracil released as acid-soluble radioactivity from poly(dA)230.poly(dT,[3H]dU)230(dT: dU = 2) after a 10-min incubation at 37 °C with an aliquot from each collected fraction diluted 5-fold in the standard mixture. From TalpaertBorl~ and Liuzzi(1982).

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Uracil r e l e a s e d (~mol)

Fig. 10. Action of uracil-DNA glycosylase from carrot cells on uracil-containing DNA-like substrates. (A) Release of uracil and appearance of AP sites as a function of time. The two standard reaction mixtures (0.2 ml) contained respectively (dA 230dT, [ 3H]dU) 230or (dA) 230" ([ 3H]dT,dU) 230with dT : dU = 15 at a concentration of 0.95 mM total nucleotides and 5/~g protein (fraction IVb). They were incubated at 37 ° C. The release of [3H]uracil from (dA)230.(dT,[3H]dU)230 (O) was estimated from the acid-soluble radioactivity; it is given as the fraction of total radioactivity. The appearance of AP sites in (dA)23o.([3H]dT,dU)23o was followed by measuring the acidsoluble fraction released after alkaline treatment (×). The acid-soluble fraction obtained without alkaline treatment was used as a control (zx). (B) Acid-soluble fraction after alkaline treatment (e) as a function of uracil released. Data are from A. From Talpaert-Borl6 and Liuzzi (1982).

We have shown that the action of u r a c i l - D N A glycosylase on uracil-containing polydeoxyribonucleotides leads to the release of uracil and to the formation of alkali-labile sites, mostly A P sites, observed f r o m the acid-soluble fraction formed by alkaline hydrolysis (Fig. 10). More recent determinations using the labelling of A P sites with [t4C]methoxyamine have shown that the n u m b e r of A P sites equals that of uracil released, This indicates that a D N A glycosylase activity can be determined either from the free base released or from the formed A P sites. We have shown that the A P endonuclease from carrot cells cuts the phosphodiester b o n d s adjacent to either apurinic sites chemically introduced in D N A or apyrimidinic sites introduced in uracil-containing polydeoxyribonucleotides under the action of u r a c i l - D N A glycosylase (Fig. 11), This suggests that the A P endonuclease is implicated in the correction of A P sites introduced by u r a c i l - D N A glycosylase in uracil-containing D N A and that these two enzymes catalyse the first steps of the base excision

?0

0'.2 0'.3 [AP sites] (,aM)

~0

lt[S 0.5

0'.4

B

100 - /

t

~

'

~

50-

©

0 '

o

o04

~

;

t'2

lAP sites] (,aM)

Fig. 11. Reaction rate of AP endodeoxyribonuclcase as a function of substrate (AP sites) concentration. Reaction rate, #, is given as the concentration of AP sites hydrolysed/min.

Inserts give Lineweaver/Burk plots of the same data. AIkylated-depurinated [14C]DNA containing 1 AP site per 16 nucleotides (A) and enzymatically depyrimidinated (dA)230. ([3H]dT,dU)230 containing 1 AP site per 19 nucleotides (B) were used. The standard incubation mixtures (0.2 ml) contained 0.1 /~g protein (fraction IVa) and were incubated at 37 o C for 2-20 min. From Talpaert-Bod6 and Liuzzi (1982).

repair process. The high content of these enzymes and their ubiquity in living cells imply that they play an important role in maintaining the genetic information. (8) Methoxyamine as an analytical tool to study repair pathways Methoxyamine has been f o u n d to be a useful reagent to analyse D N A d a m a g e and to study some enzymatic processes involved in the formation of A P sites. It allows the checking of the N-glycosyl b o n d

54 r u p t u r e u n d e r the a c t i o n of physical, c h e m i c a l or b i o c h e m i c a l agents even t h o u g h the m o d i f i e d b a s e is u n k n o w n or r e t a i n e d b y D N A . So the activity o f a D N A glycosylase whatever its specificity, either k n o w n or u n k n o w n , can b e m e a s u r e d (Liuzzi a n d Talpaert-Borl~, 1985). T h e a d d i t i o n of m e t h o x y a m i n e on the a l d e h y d e g r o u p at an A P site causes the s t a b i l i z a t i o n of the a d j a c e n t p h o s p h o e s t e r b o n d against c h e m i c a l or e n z y m a t i c r u p t u r e (Liuzzi a n d T a l p a e r t - B o r l r , 1985). Since c o n c e n t r a t i o n s of m e t h o x y a m i n e up to 50 m M d o n o t d i s t u r b the D N A glycosylase activity, it m a y be a d d e d together with the e n z y m e in o r d e r to b l o c k the A P sites a n d to p r e v e n t D N A d e g r a d a t i o n . The resulting a c c u m u l a t i o n of m e t h o x y a m i n e - r e a c t e d A P sites in a c i d - i n s o l u b l e D N A should facilitate the d e t e c t i o n of a D N A glycosylase activity in diluted or crude e n z y m a t i c p r e p a r a t i o n s . F u r t h e r m o r e , the a c c u m u l a t i o n of m e t h o x y a m i n e - r e a c t e d A P sites as well as the b l o c k i n g of the e n d o n u c l e o l y t i c r u p t u r e i n d i c a t e that the incision step occurs t h r o u g h a 2-stage incision m e c h a n i s m p r o c e e d i n g via f o r m a t i o n a n d r e p a i r of A P sites. As a c o n s e q u e n c e m e t h o x y a m i n e has b e e n p r o p o s e d as a useful reagent to s t u d y the incision step of D N A repair. T h e m e t h o d has been recently used to c o n f i r m that the cleavage of the p h o s p h o d i e s t e r b a c k b o n e at c y c l o b u t a n e p y r i m i d i n e d i m e r sites b y the U V e n d o n u c l e a s e f r o m Micrococcus luteus p r o c e e d s via a 2-step m e c h a n i s m as p r o p o s e d b y H a s e l t i n e et al. (1980) a n d R a d a n y a n d F r i e d b e r g (1980) (Liuzzi, p e r s o n a l c o m m u n i c a t i o n ) . The labelling of A P sites with r a d i o l a b e l l e d m e t h o x y a m i n e has been f o u n d to be useful also in the study of e n z y m a t i c activities involved in the excision of A P sites f r o m D N A ( G r o n d a l - Z o c c h i a n d Verly, 1985).

Acknowledgements This p u b l i c a t i o n is c o n t r i b u t i o n 2352 of the P r o g r a m m e Biology, R a d i a t i o n P r o t e c t i o n a n d M e d i c a l Research, D i r e c t o r a t e G e n e r a l X I I . for Research, Science a n d E d u c a t i o n of the C o m m i s sion of the E u r o p e a n C o m m u n i t i e s . P a r t o f the e x p e r i m e n t s were p e r f o r m e d b y M i c h e l Liuzzi w h o was a recipient of a d o c t o r a l fellowship f r o m the C o m m i s s i o n of the E u r o p e a n C o m m u n i t i e s .

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