Mutation Research, 233 (1990) 73-79 73 Elsevier

MUT 02857

Physiological properties and repair of apurinic/apyrimidinic sites and imidazole ring-opened guanines in DNA

J. Laval, S. Boiteux and T.R. O'Connor Groupe R~paration des L~sions Chimio et Radioinduites, lnstitut Gustaoe Roussy, 94805 Villejuif Cedex (France)

(Accepted 2 March 1990)

Keywords: Apurinic/apyrimidinic sites; Guanine; Imidazole ring-opened guanines in DNA

The discovery of the carcinogenic activity of N-nitrosodimethylamine by Magee and Barnes (1967) led to the realization that N-nitroso com- pounds and other alkylating agents might be an important class of highly carcinogenic compounds (for reviews, see Preussmann and Eisenbrandt, 1984; Magee and Barnes, 1967; Singer and Grun- berger, 1983; Lawley, 1984). Human contact with alkylating agents is exogenous, due to lifestyle (food, tobacco smoke, etc.) or occupation (chem- ical industry, etc.), and endogenous by in vivo formation of N-nitroso compounds. One source of endogenous N-nitroso compounds is the reaction of nitrosating agents such as nitrites with nitrosa- table amino compounds such as those present in food or drugs. These agents all modify cellular DNA and yield the same spectrum of DNA al- kylation products in vivo and in vitro (Lawley, 1976, 1984; Singer and Grunberger, 1983).

The main reaction products of alkylating agents acting on DNA are alkylpurines, phosphotriesters and, to lesser extents, alkylpyrimidines. The bio- logical effects of these adducts are not identical. Some lesions, such as 3-methylpurines (Boiteux et al., 1984; Larson et al., 1985), are potentially lethal while others, such as O6-alkylguanine or

Correspondence: Dr. J. Laval, Groupe R6paration des L6sions Chimio et Radioinduites, Institut Gustave Roussy, 94805 Vii- lejuif Cedex (France).

O4-alkylthymine (Loveless, 1969), are promuta- genic. In addition, there are also lesions, such as N7-methylguanine, that apparently present no major difficulty for the cell (Larson et al., 1985; O'Connor et al., 1988).

Some lesions arising in DNA treated with al- kylating agents are not stable and may undergo further processing yielding secondary lesions. One example of a DNA lesion which undergoes further processing is N7-methylguanine. Alkylation of the nitrogen at the 7 position of guanine may result in the formation of secondary lesions by labilizing (i) the glycosylic bond which in turn generates an AP site and (ii) the C8-N9 bond of the imidazole ring which when broken generates a formamidopyrimi- dine (Fapy) residue (Haines et al., 1962).

Alkylated bases are actively repaired in cells, but the systems involved in the repair of these damages depend on the modified base. The 3-al- kylpurines are excised by a DNA glycosylase which generates an AP site, whereas the O6-alkylguanine, O4-alkylthymine and phosphotriesters are repaired by an alkyltransferase which transfers the alkyl group to a cysteine (Lindahl et al., 1988). Sec- ondary lesions arising in DNA reacted with al- kylating agents (AP sites and Fapy residues) are also eliminated by specific DNA-repair enzymes.

In this report, we discuss the biological implica- tions of the secondary lesions which arise in DNA after treatment with alkylating agents as well as their repair in prokaryotic and mammalian cells.

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

74

Properties and repair of apurinic/apyrimidinic (AP) sites

N-Alkyl purines, which are the major lesions induced in DNA by alkylating agents, yield AP sites either by enzymatic excision by DNA glyco- sylase or by spontaneous depurination. The in vitro half-lives for depurination at neutral pH and 37 °C of N3-alkyl dA, N7-alkyl dG, and unmod- ified deoxypufines in DNA are 24, 144 and 6.4 × 106 h, respectively (Singer and Grunberger, 1983). Lawley and Brookes (1963) suggested that the mutagenic activity of alkylating agents could be related to the resultant increase in the formation of AP sites caused by these compounds. In gen- eral, electrophilic mutagens which alkylate or arylate the bases in DNA labilize the glycosidic bond (Drinkwater et al., 1980)

Previously we have shown the mutagenic poten- tial of AP sites in vitro and the preferential incor- poration of dAMP opposite to this lesion (Boiteux and Laval, 1982). It has been further demon- strated that AP sites are mutagenic intermediates in E. coli yielding mainly GC-TA base substitu- tions (for review see Loeb and Preston, 1986). Apurinic sites are also mutagenic in mammalian cells (Gentil et al., 1984).

AP sites in DNA are repaired by enzymatic cleavage of the phosphodiester backbone of the DNA strand containing the abasic deoxyribose (for recent reviews, see Weiss and Grossman, 1987; Sancar and Sancar, 1988). It has been proposed that enzymes hydrolysing the C3'-O-P bond 5' to the AP sites are AP endonucleases, whereas those breaking the C3'-O-P bond 3' to an AP site by a r-elimination mechanism are AP lyases (Bailly and Verly, 1989). These enzymes nicking DNA at AP sites have been found in virtually every organism. E. coil possesses at least 4 different AP-nicking enzymes. The xth and nfo gene prod- ucts are classified as endonucleases, whereas the nth and the fpg gene products are classified as lyases (Bailly and Verly, 1989; Bailly et al., 1989). These 4 enzymes exhibit associated activities: (i) the endonucleases are endowed with phosphatase and phosphodiesterase activities (Weiss and Grossman, 1987; Demple et al., 1986); (ii) the lyases are endowed with DNA glycosylase activi- ties acting on ring-modified pyrimidines (the nth

E.coli Map Positions of AP-Nicking Genes

10010

(Fapy DNA glycosyla se) ~ \ \

~. ~ nlh (endonucleaselll) / I (Sal t, ECOR I)

[ ~ xlh (exonuclease III) ,qfo (endonuclease IV) (EcoR I)

Fig. I. Chromosomal locat ion o f the 4 genes of E. coli known to code for proteins incising D N A at AP sites. The presence of restriction endonuclease cleavage sites in these genes for

BamHI, EcoRI, or SalI is indicated in parentheses.

gene product) or on imidazole ring-opened purines (the fpg gene product) (Sancar and Sancar, 1988; O'Connor and Laval, 1989). The location of the 4 AP-nicking activities on the E. coli chromosome is shown in Fig. 1. Mammalian AP endonucleases have been purified and characterized from sources such as yeast (Armel and Wallace, 1978), human placenta (Linsley et al., 1977; Shaper et al., 1982), HeLa cells (Kane and Linn, 1981), rat liver chro- matin (Thibodeau et al., 1980), a mouse plasma- cytoma cell line (Nes, 1980; Helland et al., 1985), and calf thymus (Henner et al., 1987). In addition to the cleavage of the phosphodiester backbone by protein molecules, simple tripeptides, such as Lys- Trp-Lys or Lys-Tyr-Lys (Pierre and Laval, 1981), acridine linked to an adenine (Constant et al., 1988) and aminoellipticine, an anticancer drug (Malvy et al., 1986), incise DNA at AP sites using a mechanism of r-elimination. Therefore, al- though inefficient in comparison to enzymatic re- pair, there is a potential for repair of AP sites by molecules catalysing r-elimination in cells. Fol- lowing incision at AP sites, the next steps in DNA repair are performed by the sequential action of various enzymes (Franklin and Lindahl, 1988).

75

Properties and repair of imidazole ring-opened purines

Methylation at the N7 position of de- oxyguanosine favors the cleavage of the glycosidic bond or the opening of the imidazole ring, which yields 2,6-diamino-4-hydroxy-5 -N-methylformam- idopyrimidine (Fapy) (Haines et al., 1962; Robins and Townsend, 1963; Chetsanga et al., .1982; Beranek et al., 1983; Boiteux et al., 1984). Fapy was prepared from 7-methylguanosine by alkali cleavage of the imidazole ring and further elimina- tion of the ribosyl residue by formic acid treat- ment. Analysis of Fapy by HPLC using a re- versed-phase column showed 2 peaks (FI and FII). Rechromatography of each isolated component indicated that they are slowly interconverted to give a 1 : 1 mixture of FI and FII (Boiteux et al., 1984). These results suggest that the FI and FII species are isomeric forms of the same molecule rather than distinct compounds. This was con- firmed by NMR studies, and the 2 species, FI and FII, were identified as rotamers E and Z respec- tively (Fig. 2). Furthermore, thermodynamic mea- surements strongly suggested that the equilibrium between the 2 isomers may be assigned to rotation around the N-methylformamido bond (Fig. 2).

Since Fapy residues exist as 2 rotamers in solu- tion, we were interested in determining the distri- bution of these isomers in duplex polynucleotides. The conditions under which chemical hydrolysis was performed are not suitable to study the con- formation of Fapy in polynucleotides. To over- come this drawback, we analyzed the products released using a large excess of pure E. coli Fapy- DNA glycosylase. The enzymatic reaction was performed for a short period of time at 5 °C in order to minimize any rotation which may occur

H'~c~O

H N ~ y N " C H 3 HN" ,~y/N ~C ~'H

NHz'N NH z

ff fll Fig. 2. Two rotamers of Fapy which are detected in solution. FI and FII are resolved by reversed-phase HPLC. FII is the

major form present in double-stranded polynucleotides.

after the excision. Under these conditions more than 95% of the Fapy residues contained in the polynucleotide were excised. Analysis of the exci- sion products by HPLC shows that 16% and 84% of the radioactive material eluted with rotamer FI and FII, respectively (Boiteux, unpublished data). This result implies that rotamer FII (Fig. 2) is the major form in double-stranded polynucleotides.

The reaction yielding Fapy occurs in vivo, since this lesion was found, as a DNA adduct, in the liver of rats treated with N,N-dimethylnitrosa- mine, or 1,2-dimethylhydrazine and in rat bladder epithelial DNA after treatment with N-methyl- nitrosourea (Beranek et al., 1983; Kadlubar et al., 1984). Several observations suggest that the im- idazole ring-opened form of N7-methylguanine might play a significant role in processes leading to mutagenesis and/or cell death by alkylating agents. In vitro DNA synthesis experiments show that Fapy residues inhibit DNA synthesis by E. coli DNA polymerase I (Boiteux and Laval, 1983; O'Connor et al., 1988). Furthermore, the termina- tion pattern of the in vitro DNA synthesis shows that E. coli DNA polymerase I stops 1 base before the Fapy residues (O'Connor et al., 1988). Since DNA synthesis terminates 1 base before N3-methyladenine lesions (Larson et al., 1985), and this lesion is a major cell-killing lesion after treatment with alkylating agents, by analogy, Fapy residues would be expected to be lethal lesions if not repaired.

An enzymatic activity for a DNA glycosylase in E. coli excising Fapy residues from DNA was first reported by Chetsanga and Lindahl in 1979 and then partially purified (Chetsanga et al., 1981). Study of this enzyme, however, was limited as a result of its low expression level in E. coli wild-type strains. Therefore, we cloned the fpg gene coding for the Fapy DNA glycosylase of E. coli on the pBR322 plasmid. The fpg gene is composed of 807 base pairs coding for a protein of 269 amino acids corresponding to the appropriate size (30 kD) and amino acid composition of the Fapy- DNA glycosylase (Boiteux et al., 1987). In ad- dition, the amino acid sequence of the first 25 amino acids is identical to that deduced from the nucleotide sequence (Boiteux et al., 1990). There- fore we conclude that the fpg gene is the struc- tural gene coding for the Fapy-DNA glycosylase

76

TABLE 1

PHYSICAL PARAMETERS OF THE FPG PROTEIN

Molar absorption coefficient at 280 n m

- Calculated 3.1 × 104 1/(mol-cm) - Experimentally determined 3.9 )< 104 1/(mol-cm)

Stokes' radius, gel filtration chromatography 2.5 n m

Molecular weight - Calculated from nucleotide

sequence 30.2 kD - Sodium dodecyl sulfate gel

electrophoresis 31 + 1 kD - Gel filtration chromatography 30 + 2 kD

Isoelectric point - Calculated 8.6 - Isoelectric focusing gel

electrophoresis 8.5 - FPLC 7.9

Denaturation: za H( - E D T A / + EDTA) 83/109 kca l /mol A S( - E D T A / + EDTA) 250/350 ca l /deg-mol

in E. coli and we renamed it the FPG protein (Boiteux et al., 1990). The fpg gene was further subcloned in the pUC18 and pUC19 plasmids to yield pFGP50 and 60, respectively. Despite the 60-fold overproducer character of the cells harbor- ing the pFPG60 plasmid, the FPG protein did not exceed 1% of the total soluble proteins (Boiteux et al., 1987). This relatively low expression of the gene was mainly due to the inefficiency of the transcription from the lac promoter. Deletion of a palindromic sequence upstream from the fpg pro- moter increased the level of the FPG protein to 5-10% of the total soluble cellular proteins (O'Connor et al., 1988; Boiteux et al., 1990).

Using the cloned fpg gene, the FPG protein was overproduced and purified to homogeneity. The physical parameters of the protein were mea- sured. The results are summarized in Table 1. This protein is a small molecule of 30.2 kD with 1 zinc per molecule and acts as a globular monomer (Boiteux et at., 1990). During the purification of the FPG protein, the Fapy-DNA glycosylase ac- tivity copurifies with an AP-nicking activity using chromatographic separations based on ion ex- change, molecular weight exclusion, affinity, and

hydrophobicity (O'Connor and Laval, 1989). Fur- thermore, the sequencing from the N terminal of the 25 first amino acids from the purified FPG protein yielded a single amino acid sequence cor- responding to the amino acid sequence of the FPG protein (Boiteux et al., 1990). These results lead us to conclude that the FPG protein is en- dowed with at least 2 different enzymatic activi- ties: a Fapy-DNA glycosylase and an AP-nicking activity.

The mechanism of DNA-strand nicking at AP sites by the FPG protein was investigated using 2 different approaches. In one set of experiments DNA incised at AP sites by the FPG protein was used with or without pretreatment with phos- phatase as a substrate either for DNA polymerase I or for DNA polynucleotide kinase (O'Connor and Laval, 1989). In another set of experiments, a synthetic oligonucleotide containing an AP site at a precise location was labeled either at the 5' or at the 3' end hybridized with the complementary strand and used as a substrate for the FPG pro- tein. The products of the second set of experi- ments were analyzed using denaturing poly- acrylamide gel electrophoresis (Bailly et al., 1989). Both experiments show that the FPG protein cleaves both phosphodiester bonds 5' and 3' to the AP site (Fig. 3). Using DNA containing a deoxyribose radioactively labeled at the deoxyri-

B B F" B B B

. . . . b 4 4 . . . .

Jr Fapy-DNA Glycosylase

B B B B B

. . . . 4 4 4 . . . .

2 '~COH ,C ~'0 Labelled \H Deoxyrlbose

Derivative

3 F ~ Fapy base

Fig. 3. Products of base excision from D N A using the FPG protein of E. coli. Only a single strand of the D N A is shown for simplicity. The products are (1) D N A with a gap limited by 3' and 5 ' ends on the D N A strand containing the abasic site, (2) an unsaturated, organic soluble product resulting from the

f l -~-el iminat ions, and (3) the Fapy base.

bose of the AP site we observed that the FPG protein released an uncharged radiolabeled mole- cnle, soluble in organic solvents which is adsorbed on Norite (Fig. 3) (Bailly et al., 1989). Reduced AP sites are not substrates for the AP lyase activ- ity of the FPG protein. All these data are con- sistent with a fl-elimination followed by a 8- elimination. The products of the reaction cata- lysed by the FPG protein for one strand of DNA are shown in Fig. 3.

The DNA glycosylase activity associated with the FPG protein exhibits a broad substrate specificity since it excises the imidazole ring- opened form of adenine, a lesion occurring after X-irradiation (Breimer, 1984), the imidazole ring- opened form of guanine modified at the N7 posi- tion by methylating agents (see above), chloroeth- ylating agents (Laval et al., 1990), aflatoxin B 1 (Chetsanga and Frenette, 1983), and phosphor- amide mustard (Chetsanga et al., 1982). All these formamidopyrimidine derivatives have in common an imidazole ring opened between the C8 and the N9 of guanine residues. Moreover, the FPG pro- tein also excises imidazole ring-opened guanines between the N7 and C8 of the guanine such as the imidazole ring-opened form of N-(deoxyguanosin- 8-yl)-2-aminofluorene (Boiteux et al., 1989). De- spite the fact that the FPG protein will excise a variety of lesions with ring-opened purines, this protein will not remove Fapy lesions from poly- nucleotides in a Z-DNA conformation (Lagravtre, 1984).

Conclusions

Secondary lesions arising in DNA treated with alkylating agents have deleterious effects on the cells, if not repaired. Apurinic sites are (i) cell-kill- ing lesions since E. coli mutants defective in the 2 major AP endonucleases are highly sensitive to genotoxic treatments leading to the formation of AP sites in DNA (Cunningham et al., 1986), (ii) promutagenic lesions which certainly contribute to the mutagenesis and carcinogenesis by alkylating agents (Loeb and Preston, 1986).

The biological effect of imidazole ring-opened purines remains speculative. (i) The E. coil mutant defective in FPG protein does not exhibit an unusual sensitivity to DNA-damaging agents be-

77

lieved to generate Fapy residues (Boiteux and Huisman, 1989); (ii) the occurrence of imidazole ring-opened products in vivo was only established after treatment with aflatoxin B 1 (Essigman et al., 1983). Since the FPG protein excises many differ- ent adducts the lack of sensitivity of the E. coli defective mutant might be due to the fact that the physiological substrate has not been identified. Alternatively some Fapy residues might be re- paired by another pathway than the FPG protein. Another role of the FPG protein may be to re- move minor lesions induced by different treat- ments including alkylation, oxidation, and radia- tion. However, the fact that Fapy-DNA glyco- sylase is highly conserved in prokaryotes (Boiteux et al., 1990) and eukaryotes suggests that this enzyme may contribute to the maintenance of genetic information.

Acknowledgements

We thank Claudine Lagrav&e d'Herin and Patricia Auffret van der Kemp for their excellent technical assistance. This work and T.R.O. were supported by the CNRS, INSERM, the Associa- tion pour la Recherche sur le Cancer, the Ligue National Franqalse contre le Cancer and the Fondation pour la Recherche M6dicale.

References

Armel, P.R., and S.S. Wallace (1978) Apurinic endonucleases from Saccharomyces cereoisiae, Nucleic Acids Res., 5, 3347-3356.

Bailly, V., and W.G. Verly (1989) AP-endonucleases and AP lyases, Nucleic Acids Res., 17, 3617-3618.

Bailly, V., W.G. Verly, T.R. O'Connor and J. Laval (1989) Mechanism of DNA strand nicking at apurinie/apyrimi- dinic sites by Escherichia coli (formamidopyrimidine) DNA glycosylase, Biochem. J., 262, 581-589.

Beranek, D.T., C.C. Weis, F.E. Evans, C.J. Chetsanga and F.F. Kadlubar (1983) Identification of NS-methyl-NS-formyl- 2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,- Nq-dimethylnitrosamine or 1,2-dimetliylhydrazine, Bio- chem. Biophys. Res. Commun., 110, 625-631.

Boiteux, S., and O. Huisman (1989) Isolation of a formami- dopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12, Mol. Gen. Genet., 215, 300-305.

Boiteux, S., and J. Laval (1982) Coding properties of poly(de- oxy-cytidilic acid) templates containing uracil or apyrimi- dinic sites, Biochemistry 21, 6746-6751.

78

Boiteux, S., and J. Laval (1983) Imidazole ring-opened guanine: an inhibitor of DNA synthesis, Biochem. Biophys. Res. Commun., 110, 625-631.

Boiteux, S., J. Belleney, B.P. Roques and J. Laval (1984a) Two rotameric forms of ring-open 7-methylguanine are present in alkylated polynucleotides, Nucleic Acids Res., 6, 3673- 3683.

Boiteux, S., O. Huisman and J. Laval (1984b) 3-Methyladenine residues in DNA induce the SOS function sfia in Escherichia coli, EMBO J., 3, 2569-2573.

Boiteux, S., T.R. O'Connor and J. Laval (1987) Formami- dopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduc- tion of the protein, EMBO J., 6, 3177-3183.

Boiteux, S., M. Bichara, R.P.P. Fuchs and J. Laval (1989) Excision of the imidazole ring-opened form of N-2- aminofluorene-C(8)-guanine adduct in poly(dG-dC) by E. coli formamidopyrimidine-DNA glycosylase, Carcinogene- sis, 10, 1905-1909.

Boiteux, S., T.R. O'Connor, F. Lederer, A. Gouyette and J. Laval (1990) Homogeneous FPG protein: a DNA glyco- sylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites, J. Biol. Chem., 265, 3916-3922.

Boiteux, S., M.A. Lemaire and J. Laval (1990) Conservation of formamidopyrimidine DNA glycosylase in prokaryotes, in: S. Wallace and R. Painter (Eds.), UCLA Symposia on Molecular and Cellular Biology: Ionizing Radiation Damage to DNA, Liss, New York, in press.

Breimer, L. (1984) Enzymatic excision from y-irradiated poly- nucleotides of adenine residues whose imidazole rings have been ruptured, Nucleic Acids Res., 12, 6359-6367.

Chetsanga, C.J., and G.P. Frenette (1983) Excision of aflatoxin Bl-imidazole ring-opened guanine adducts from DNA by formamidopyrimidine DNA glycosylase, Carcinogenesis, 4, 997-1000.

Chetsanga, C.J., and T. Lindahl (1979) Release of 7-methyl- guanine whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli, Nucleic Acids Res., 6, 3673-3683.

Chetsanga, C.J., M. Lozon, C. Makaroff and L. Savage (1981) Purification and characterization of Escherichia coli for- mamidopyrimidine-DNA glycosylase that excises damaged 7-methylguanine from deoxyribonucleic acid, Biochemistry, 20, 5201-5207.

Chetsanga, C.J., G. Polidori and M. Mainwaring (1982a) Anal- ysis and excision of ring-opened phosphoramide mustard deoxyguanosine adducts in DNA, Cancer Res., 42, 2626- 2631.

Chetsanga, C.J., B. Bearie and C. Makaroff (1982b) Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analy- sis of the resulting pyrimidine derivatives, Chem.-Biol. In- teract., 41, 217-233.

Constant, J.F., T.R. O'Connor, J. Lhomme and J. Laval (1988) 9-[(10-(Aden-9-yl)-4,8-diazadecyl)amino]-6-chloro-2-meth- oxyacridine incises DNA at apurinic sites, Nucleic Acids Res., 16, 2691-2703.

Cunningham, R.P., S.M. Saporito, S.G. Spitzer and B. Weiss

(1986) Endonuclease IV (nfo) of Escherichia coli, J. Bacteriol., 168, 1120-1127.

Demple, B., A. Johnson and D. Fung (1986) Exonuclease III and endonuclease IV remove 3' blocks from DNA synthe- sis primers in H202 damaged Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 83, 7731-7735.

Drinkwater, N.R., E.C. Miller and J.A. Miller (1980) Estima- tion of apurinic/apyrimidinic sites and phosphotriesters in deoxyribonucleic acid treated with electrophilic carcinogens and mutagens, Biochemistry, 19, 5087-5092.

Essigman, J.M., C.L. Green, R.C. Croy, K.W. Fowler, G.H. Buchi and G.M. Wogan (1983) Interaction of aflatoxin B1 and alkylating agents with DNA: structural and functional studies, Cold Spring Harbor Symp. Quant. Biol., 47, 327- 337.

Franklin, W.A., and T. Lindahl (1988) DNA deoxyribophos- phodiesterase, EMBO J., 7, 3617-3622.

Gentil, A., A. Margot and A. Sarasin (1984) Apurinic sites cause mutations in simian virus 40, Mutation Res., 129, 141-147.

Haines, J.A., C.B. Reese and J. Lord Todd (1962) The methyl- ation of guanosine and related compounds with di- azomethane, J. Chem. Soc., 5281-5288.

Helland, D.E., A.J. Raae, P. Fadnes and P. Kleppe (1985) Properties of a DNA repair endonuclease from mouse plasmacytoma cells, Eur. J. Biochem., 148, 471-477.

Henner, D.W., N.P. Kiker, T.J. Jorgensen and J.N. Munck (1987) Purification and amino acid sequence of an apurinic/apyrimidinic endonuclease from calf thymus, Nucleic Acids Res., 15, 5529-5544.

Kadhibar, F.F., D.T. Beranek, C.C. Weiss, F.E. Evans, R. Cox and C.C. Irving (1984) Characterization of ring opened 7-methylguanine and its persistence in rat bladder epithelial DNA after treatment with the carcinogen N-methyl- nitrosourea, Carcinogenesis, 5, 587-592.

Kane, C.M., and S. Linn (1981) Purification and characteriza- tion of an apurinic/apyrimidinic endonuclease from HeLa cells, J. Biol. Chem., 256, 3405-3414.

Lagravtre, C., B. Malfoy, M. Leng and J. Laval (1984) Ring opened alkylating guanine is not repaired in Z-DNA, Na- ture (London), 310, 798-800.

Larson, K., J. Sham, R. Shenkar and B. Strauss (1985) Methyl- ation-induced blocks to in vitro DNA replication, Mutation Res., 150, 77-84.

Laval, J., F. Lopez, J. Madelmont, C. Godtneche, G. Meyniel, Y. Habraken, T.R. O'Connor and S. Boiteux (1990) Exci- sion of imidazole ring-opened N7-hydroxyethylguanine from chloroethylnitrosourea-treated D N A by E. coli for- mamidopyrimidine-DNA glycosylase, in: H. Bartsch and J.C. Chen (Eds.), N-Nitroso Compounds, Mycotoxins, and Tobacco Smoke: Relevance to Human Cancer, Sci. Publ. 105, IARC, Lyon, in press.

Lawley, P.D. (1976) Methylation of DNA by carcinogens: some applications of chemical analytical methods in screen- ing tests in chemical carcinogenesis, Sci. PUbl. 12, IARC, Lyon, pp. 181-210.

Lawley, P.D. (1984) Carcinogenesis by alkylating agents, in: C.E. Searle (Ed.), Chemical Carcinogens, 2nd edn., ACS

monograph 182, American Chemical Society, Washington DC, pp. 325-484.

Lawley, P.D., and P. Brookes (1983) Further studies on the alkylation of nucleic acids and their constituent nucleo- tides, Biochem. J., 89, 127-138.

Lindahl, T., B. Sedgwick, M. Sekiguchi and Y. Nakabeppu (1988) Regulation and expression of the adaptive response to alkylating agents, Armu. Rev. Biochem., 57, 133-157.

Linsley, W.S., E.E. Penhoet and S. Linn (1977) Human endo- nuclease specific for apurinic/apyrimidinic sites in DNA, J. Biol. Chem., 252, 1235-1242.

Loeb, L.A., and B.D. Preston (1986) Mutagenesis by apurinic/apyrimidinic sites, Annu. Rev. Genet., 20, 201- 230.

Loveless, A. (1969) Possible relevance of O6-alkylation of deoxyguanosine to mutagenicity of nitrosamines and nitrosamides, Nature (London), 223, 206-208.

Magee, P.N., and J.M. Barnes (1967) Carcinogenic nitroso compounds, Adv. Cancer Res., 10, 163-246.

Malvy, C., P. Pr6vost, C. Gansser, C. Viel and C. Paoletti (1986) Efficient breakage of DNA apurinic sites by the indole-amine related 9-amino-eUipticine, Chem.-Biol. Inter- act., 57, 41-53.

Nes, I.F. (1980) Purification and characterization of an endo- nuclease specific for apurinic sites in DNA from a perma- nently established mouse plasmacytoma cell line, Nucleic Acids Res., 8, 1575-1589.

O'Connor, T.R., and J. Laval (1989) Physical association of the 2,6-diamino-4-hydroxy-5N- formamidopyrimidine- DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites, Proc. Natl. Acad. Sci. (U.S.A.), 86, 5222-5228.

O'Connor, T.R., S. Boiteux and J. Laval (1988) Ring-opened

79

7-methylguanine residues in DNA are a block to in vitro DNA synthesis, Nucleic Acids Res., 16, 5879-5894.

O'Connor, T.R., S. Boitenx and J. Laval (1989) Repair of imidazole ring-opened purines in DNA: overproduction of the formamidopyridimidine-DNA glycosylase of Escheri- chia coli using plasmids containing the fpg+ gene, Ann. Ist. Super. Sanith, 25, 27-32.

Pierre, J., and J. Laval (1981) Specific nicking of DNA at apurinic sites by peptides containing aromatic residues, J. Biol. Chem., 256, 10217-10220.

Prenssmann, R., and G. Eisenbrand (1984) N-nitroso carcino- gens in the environment, In: C.E. Searle (Ed.), Chemical Carcinogens, ACS Monograph 182, American Chemical Society, Washington DC, pp. 829-868.

Robins, R.K., and L.B. Townsend (1963) Ring cleavage of purinenucleosides to yield possible biogenetic precursors of pteridines and riboflavin, J. Am. Chem. Soc., 83, 242.

Sancar, A., and G.B. Sancar (1988) DNA repair enzymes, Annu. Rev. Biochem., 57, 29-67.

Shaper, N.L., R.H. Grafstrom and L. Grossman (1982) Human placental apurinic/apyrimidinic endonuclease: its isolation and characterization, J. Biol. Chem., 257, 13455-13458.

Singer, B., and D. Gnmberger (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York.

Thibodeau, L., S. Bricteux and W.G. Verly (1980) Purification and properties of the major apurinic/apyrimidinic endode- oxyribonuclease of rat liver chromatin, Eur. J. Biochem., 110, 379-385.

Weiss, B., and L. Grossman (1987) Phosphodiesterases in- volved in DNA repair, in: Advances in Enzymology, John Wiley and Sons, New York, pp. 1-34.