Translesional Synthesis on DNA Templates Containing a Single Abasic Site

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase α. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase α in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells. Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase α. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase α in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells. Abasic sites in DNA arise spontaneously by hydrolysis, a process that can be accelerated by modification of purine bases (1Lindahl T. Nyberg B. Biochemistry. 1972; 11: 3610-3618Google Scholar) and by the catalytic action of N-glycosylases that remove damaged bases from DNA (2Loeb L.A. Preston B.D. Annu. Rev. Genet. 1986; 20: 201-230Google Scholar, 3Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Google Scholar, 4Weiss B. Grossman L. Adv. Enzymol. Relat. Areas Mol. Biol. 1987; 60: 1-34Google Scholar). The natural abasic site (Ab) 1The abbreviations used are: Ab, natural apurinic/apyridiminic (abasic) site; F, tetrahydrofuran; Re, reduced form of the natural abasic site (deoxyribitol); pol, DNA polymerase; HPLC, high performance liquid chromatography. exists as an equilibrium mixture of the cyclic hemiacetal and open chain aldehyde forms of 2′-deoxyribose (see Fig. 1) and is subject to β-elimination. This chemical reaction leads to strand scission (5Jones A.S Mian A.M Walker R.T J. Chem. Soc.C. 1968; : 2042-2044Google Scholar); for that reason, structural analogs of deoxyribose have often been used to explore biological properties of abasic sites in DNA (6Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Google Scholar, 7Sagher D. Strauss B. Nucleic Acids Res. 1985; 13: 4285-4298Google Scholar, 8Randall S.K. Eritja R. Kaplan B.E. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 6864-6870Google Scholar, 9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar, 10Millican T.A. Mock G.A. Chauncey M.A. Patel T.P. Eaton M.A.W. Gunning J. Cutbush S.D. Neidele S. Mann J. Nucleic Acids Res. 1984; 12: 7435-7453Google Scholar). Abasic site analogs include deoxyribitol, a model for the open chain form of the sugar (6Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Google Scholar, 7Sagher D. Strauss B. Nucleic Acids Res. 1985; 13: 4285-4298Google Scholar), and tetrahydrofuran, an isosteric and isoelectronic analog of deoxyribose (8Randall S.K. Eritja R. Kaplan B.E. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 6864-6870Google Scholar, 9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar) that is cleaved by type II AP endonucleases, but is not subject to β-elimination (9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar). The miscoding properties of natural and synthetic abasic sites have been investigated under a variety of experimental conditions using randomly or site-specifically modified DNA (6Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Google Scholar, 7Sagher D. Strauss B. Nucleic Acids Res. 1985; 13: 4285-4298Google Scholar, 11Hevronoi D. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5046-5050Google Scholar, 12Kunkel T.A. Schaaper R.M. Loeb L.A. Biochemistry. 1983; 22: 2378-2384Google Scholar, 13Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Google Scholar, 14Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1494-1498Google Scholar, 15Lawrence C.W. Borden A. Banerjee S.K. LeClerc J.E. Nucleic Acids Res. 1990; 18: 2153-2157Google Scholar). The relative frequency of base incorporation opposite abasic sites and of chain extension from the 3′-primer terminus has also been reported (8Randall S.K. Eritja R. Kaplan B.E. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 6864-6870Google Scholar, 9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar). While natural and synthetic abasic sites are structurally similar, their mutagenic potential has not been compared under the same experimental conditions. In Escherichia coli, synthesis past abasic sites in vitro (6Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Google Scholar, 7Sagher D. Strauss B. Nucleic Acids Res. 1985; 13: 4285-4298Google Scholar, 8Randall S.K. Eritja R. Kaplan B.E. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 6864-6870Google Scholar, 9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar, 11Hevronoi D. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5046-5050Google Scholar, 12Kunkel T.A. Schaaper R.M. Loeb L.A. Biochemistry. 1983; 22: 2378-2384Google Scholar, 13Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Google Scholar) and in vivo (14Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1494-1498Google Scholar, 15Lawrence C.W. Borden A. Banerjee S.K. LeClerc J.E. Nucleic Acids Res. 1990; 18: 2153-2157Google Scholar) is accompanied by preferential incorporation of dAMP opposite the lesion, a phenomenon known as the “A rule” (16Strauss B. Bioassays. 1991; 13: 79-84Google Scholar). In eukaryotes, the presence of abasic sites in DNA is associated with different mutational spectra. For example, dAMP, dCMP, and dTMP were inserted at similar frequencies opposite a natural abasic site when a plasmid vector containing this lesion was allowed to replicate in simian kidney (COS) cells (17Gentil A. Renault G. Madzak C. Margot A. Cabral-Neto J.B. Vasseur J.J. Rayner B. Sarasin A. Biochem. Biophys. Res. Commun. 1990; 173: 704-710Google Scholar, 18Gentil A. Cabral-Neto J.B. Mariage-Samson R. Margot A. Imbach J.L. Rayner B. Sarasin A. J. Mol. Biol. 1992; 227: 981-984Google Scholar, 19Cabral Neto J.B. Gentil A. Cabral R.E.C. Sarasin A. J. Biol. Chem. 1992; 267: 19718-19723Google Scholar, 20Cabral Neto J.B. Cabral R.E.C. Margot A. Le Page F. Sarasin A. Gentil A. J. Mol. Biol. 1994; 240: 416-420Google Scholar). In another study in COS cells, preferential incorporation of dAMP was observed opposite the tetrahydrofuran moiety, accompanied by a small number of deletions (21Takeshita M. Eisenberg W. Nucleic Acids Res. 1994; 22: 1897-1902Google Scholar). In human lymphoblastoid cells, dGMP was incorporated preferentially opposite natural abasic sites (22Klinedinst D.K. Drinkwater N.R. Mol. Carcinog. 1992; 6: 32-42Google Scholar). In AP endonuclease-deficient strains of yeast, the frequency of A:T → C:G events increased (23Kunz B.A. Henson E.S. Roche H. Ramotar D. Nunoshiba T. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8165-8169Google Scholar); in another yeast system, dCMP was predominantly incorporated opposite the lesion (24Gibbs P.E. Lawrence C.W. J. Mol. Biol. 1995; 251: 229-236Google Scholar). In this report, we used a prokaryotic and a eukaryotic DNA polymerase and DNA templates containing a single abasic site to explore the mechanistic basis underlying mutagenesis at abasic sites in DNA. Recently, one of us (S. S.) developed a method by which site-specifically modified oligodeoxynucleotides could be used to quantify all base substitutions and deletions occurring during DNA synthesis in vitro (25Shibutani S. Chem. Res. Toxicol. 1993; 6: 625-629Google Scholar). Combined with steady-state kinetic analysis (26Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Google Scholar, 27Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar), this experimental system is used to compare the miscoding properties of a natural abasic site and its analogs in reactions catalyzed by the Klenow fragment of E. coli DNA polymerase I or by calf thymus DNA polymerase α. Our results indicate that the natural abasic site resembles closely the tetrahydrofuran moiety with respect to its miscoding properties. These lesions, which exist primarily or exclusively in a ring-closed conformation, preferentially incorporate dAMP opposite the lesion. In contrast, deoxyribitol, which serves as a model for the open chain (minor) form of the natural abasic site, blocks DNA synthesis and promotes the sequence-dependent formation of 1- and 2-base deletions.In vitro, both pol α and the Klenow fragment operate according to the tenets of the A rule. Organic chemicals used for the synthesis of oligodeoxynucleotides were supplied by Aldrich. γ-32PATP (specific activity > 6000 Ci/mmol) was obtained from Amersham Corp. Cloned exo+ (17,400 units/mg) and exo− (21,200 units/mg) Klenow fragments of E. coli polymerase I (1 unit of enzyme catalyzes the incorporation of 1 nmol of total nucleotide into form in at and (1 were from U. S. Corp. DNA pol I and were from calf thymus DNA pol α units/mg) (1 unit of enzyme catalyzes the incorporation of nmol of nucleotide in at using as was from was from and I was from and all were from A with a was used to and modified and were synthesized by using an DNA containing a single or tetrahydrofuran at and in were synthesized as (9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar). containing a single natural abasic site (Ab) were by of an containing a single 1 or in of at for T. J. Biochem. J. 1989; 262: Scholar). The reaction mixture was to in a and to The containing an site was from the a using a of containing an of and a of S. R. Johnson F. Grollman A.P. 1991; 12: Scholar). containing the was using a The was three with 1 of at of the natural site was at in was in three J. J. J. Biol. Chem. Scholar). were with a and to containing the reduced form of the natural abasic site were as was using of a were for at with of in a reaction containing and 1 The was from the a using a of containing an of and of were by in the presence of and at the by with in the presence of γ-32PATP F. J. A Scholar). were to containing The of the was by using of are the of and or T. in a are the of and or T. The of the natural abasic site was as was cleaved at the of the lesion with 1 In contrast, and of were in when at and in reported (9Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Google Scholar), containing and were in when at C. This reaction was as in from this (25Shibutani S. Chem. Res. Toxicol. 1993; 6: 625-629Google Scholar, S. M. Grollman A.P. 1991; Scholar, S. Grollman A.P. Biochemistry. 1993; Scholar). or or a containing or a single Ab, Re, or at the was to of a or or primer or extension was in a containing and an or modified DNA with or Klenow fragment with or → activity was at or for 1 in containing and extension reactions with pol α were at in of containing and using a modified to a primer were to analysis by or with in the and in the (25Shibutani S. Chem. Res. Toxicol. 1993; 6: 625-629Google Scholar). of the were by was in a The for reaction products was of the to nucleotide insertion and chain extension were under conditions similar to for the primer extension S. M. Grollman A.P. 1991; Scholar, S. Grollman A.P. Biochemistry. 1993; Scholar). containing of exo+ or exo− Klenow fragment were at for in of containing of DNA Ab, Re, or with of to nucleotide insertion or with or to chain extension as by (26Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Google Scholar, 27Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar). insertion was in reactions using of exo+ Klenow fragment for for and for and and for for and of exo− Klenow fragment for for and and for and of extension were in reactions using of exo+ Klenow fragment for and for for and of exo− Klenow fragment for for and for for a DNA the frequency of incorporation at the blunt end of the was in reactions using of exo+ Klenow fragment for were for at in the presence of and to a in the presence of were by and as for the and the of the reaction were obtained from The of to enzyme is at similar were reported by M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: Scholar). than of the primer is extended under the steady-state conditions used in S. H. Goodman M.F. J. Biol. Chem. 1992; 267: Scholar). reported an of to of nucleotide insertion and chain extension were relative to according to by L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Google Scholar, 27Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar), with as a or Ab, Re, or F. The of the natural and synthetic abasic sites used in these are in extension catalyzed by the exo− Klenow fragment of DNA polymerase were in the presence of The reaction by a mixture of oligodeoxynucleotides containing and 1- or 2-base deletions (25Shibutani S. Chem. Res. Toxicol. 1993; 6: 625-629Google Scholar), were by as in 1 and DNA synthesis an to the incorporation of dCMP of the opposite at with deletions. an was used as dAMP was preferentially incorporated opposite the 2-base deletions were also results were obtained using an In contrast, of primer extension an containing Re, the of fully extended containing a 2-base dAMP incorporation opposite the lesion and deletions The of these products increased and was the of enzyme used in the reaction not of dGMP and dTMP opposite abasic sites was not In the presence of blunt end extension was observed in and of primer extension in reactions catalyzed by the exo− Klenow fragment templates containing abasic not an or abasic with a primer extension reactions were at using of exo− Klenow fragment for the containing and for that containing abasic sites as under and the of the fully extended containing and 1- and 2-base deletions opposite the lesion from the The in the relative of observed at and to at not in a an or abasic with a primer extension reactions were at using of exo− Klenow fragment for the containing and for that containing abasic sites as under and the of the fully extended containing and 1- and 2-base deletions opposite the lesion from the The in the relative of observed at and to at The of the base incorporated and the of deletions during translesional synthesis templates were by sequence analysis W. in Enzymol. Scholar). The fully extended reaction products at and dAMP incorporation opposite and a 2-base opposite the lesion, not results were obtained by sequence analysis of fully extended products or templates not for nucleotide insertion opposite Ab, Re, or and for chain extension from the 3′-primer terminus were under steady-state conditions as S. Grollman A.P. J. Biol. Chem. 1993; Scholar, S. Johnson F. Grollman A.P. Biochemistry. 1993; Scholar). The exo+ Klenow fragment → and the exo− Klenow fragment both → and → in the relative insertion frequency opposite the abasic site for the exo+ Klenow fragment the order dAMP > dGMP > dCMP > for dAMP incorporation opposite abasic sites was than for dAMP opposite and than for dGMP opposite Ab, Re, or F. dAMP opposite was than for dAMP opposite or could be for the and a used to the frequency of translesional synthesis G. Goodman M.F. B. Biochemistry. 1991; Scholar), was for than for for blunt end extension was S. Goodman M.F. J. Biol. Chem. 1995; Scholar), dAMP was inserted exclusively at a than that for dAMP incorporated opposite Ab, Re, or for nucleotide insertion and chain extension reaction catalyzed by the exo+ Klenow fragment of DNA polymerase not of insertion and extension reactions were as under of nucleotide insertion and chain extension were by the or modified blunt end not in a of insertion and extension reactions were as under of nucleotide insertion and chain extension were by the or modified blunt end the exo− Klenow fragment for dAMP opposite Ab, Re, or was than for for dAMP opposite was than for dAMP opposite Re, but less than for dAMP opposite F. from 3′-primer containing was than from containing and less than from synthesis past was to be than synthesis past and than past for nucleotide insertion and chain extension reaction catalyzed by the exo− Klenow fragment of DNA polymerase frequency of base insertion and chain extension was as under the to in a The frequency of base insertion and chain extension was as under the to extension reactions were using a modified in which is positioned to extended products this dAMP or a opposite the 2-base deletions were not and and at extended products in which dGMP and dAMP have been incorporated opposite the lesion, The and also extended of in Fig. were and compared in obtained in with oligodeoxynucleotides in which is positioned to this fully extended products containing 2-base deletions were The of the inserted dAMP and 1- and 2-base deletions were by sequence analysis not pol α was used to primer translesional synthesis past and and of fully extended products containing dAMP opposite the lesion and and of 2-base deletions 1 and In contrast, primer extension the was opposite and 1 base the lesion The of dAMP incorporation and 2-base deletions and templates increased blunt end addition reactions were observed and Fig. and 2-base deletions were observed opposite and and 2-base deletions opposite and dCMP were not incorporated opposite properties by the natural and abasic the and templates with a primer extension reactions were at for 1 using or of pol α as under 1 and the oligodeoxynucleotides containing and opposite the lesion and 1- and 2-base deletions as in the to Fig. In this the mutagenic potential of Ab, a reduced form of this lesion and an isosteric synthetic analog of deoxyribose have been compared using an experimental system (25Shibutani S. Chem. Res. Toxicol. 1993; 6: 625-629Google Scholar) that us to and quantify all base substitutions and 1- and 2-base deletions in the exo− Klenow fragment of DNA pol I was used to primer extension in the presence of all three types of abasic sites dAMP 2-base and a small number of deletions. containing or predominantly incorporated dAMP opposite the lesion, templates containing formation of 2-base deletions. In exists as an equilibrium mixture of primarily of the and of accompanied by a small of the aldehyde is a structural analog of the cyclic hemiacetal form of Ab, is an analog of the open chain aldehyde three translesional synthesis and formation of and translesional synthesis than Re, that the open chain form of is a lesion. extension were in at or for 1 kinetic were in the same at for We that of the was cleaved by β-elimination in 1 under these conditions. not the kinetic reported in this study for the natural abasic site. the exo+ Klenow fragment was used to DNA synthesis, chain extension was opposite and 1 base Ab, F, and not the order dAMP > dGMP dTMP and dCMP, for or was much than for or in with the that the of translesional synthesis opposite the lesion The relative for these are similar to with but the of translesional synthesis was of for exo+ than for the → of DNA pol I to the of synthesis at abasic when polymerase the polymerase of E. coli, was used in a similar primer extension chain extension was 1 base the abasic site, that the → of this enzyme can remove a inserted opposite the lesion. M. and A. The reported for with exo+ could be by the → activity of this enzyme S. Goodman M.F. J. Biol. Chem. 1995; Scholar). in the relative of of dAMP and positioned opposite at the 3′-primer terminus are at H. H. S. H. M. Nucleic Acids Res. 1995; Scholar). for be relative and from the with exo+ DNA polymerases nucleotide addition at the blunt end of DNA J. Mol. Biol. 1987; Scholar). The frequency of base addition at this was dAMP addition was This that preferential incorporation of dAMP is an of DNA polymerase (16Strauss B. Bioassays. 1991; 13: 79-84Google for the addition reaction was of than for dAMP insertion opposite abasic 1- and 2-base deletions opposite abasic sites were when was used in the for primer with S. Grollman A.P. J. Biol. Chem. 1993; Scholar) and S. Grollman A.P. Biochemistry. 1993; Scholar), we have a model to for deletions. This model that extension of a inserted is the inserted at the 3′-primer terminus preferentially form a with a base positioned to the lesion S. Grollman A.P. J. Biol. Chem. 1993; Scholar). In with the exo− Klenow primer extension the sequence was opposite the abasic site for opposite the lesion the order dAMP > dGMP > dCMP and dTMP. when dGMP is inserted opposite the abasic site, the inserted nucleotide can with to the lesion to form a as in Fig. A. dAMP and the in the primer could with to the lesion to form a 2-base both products were Our kinetic indicate that is a lesion than or F, and as for the sequence used in this was in promoting 2-base deletions than were ring-closed forms of the abasic site. the base to in the was from to the frequency of deletions increased was dAMP is inserted than dGMP opposite the lesion, and deletions are by preferential and to the lesion This is with the for deletions to in the presence of S. Grollman A.P. J. Biol. Chem. 1993; Scholar). dAMP incorporation increased the number of 1- and 2-base deletions increased This the for the to a as in model S. Grollman A.P. J. Biol. Chem. 1993; Scholar), of the relative number of base substitutions and deletions during translesional The frequency of nucleotide insertion opposite synthetic abasic sites and of chain extension from the 3′-primer terminus has been for pol α (26Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Google Scholar, 27Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar). A was insertion (26Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Google Scholar). The study is the using a mammalian DNA polymerase and a natural abasic site in which of and events have been site-specifically in pol α incorporation of dAMP opposite and and 1- and 2-base of fully extended products were templates containing than containing F. In contrast, primer extension an was opposite and 1 base the lesion. The of translesional synthesis past was than that templates containing F. the miscoding properties of pol α and exo− in vitro with respect to and are pol another mammalian was used in an primer extension preferential incorporation of dAMP opposite also was Proc. Natl. Acad. Sci. in U. S. Scholar). We have mutagenesis a number of DNA and in which nucleotide by primer extension analysis and steady-state was compared in the same sequence with mutational in in and mammalian cells M. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar, M. W. Johnson F. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: Scholar). In the preferentially incorporated by prokaryotic or eukaryotic DNA polymerases in vitro was in the mutational of the lesion as observed in cells. The miscoding properties of the natural abasic site, in pol α and pol predict dAMP incorporation at abasic sites in mammalian cells, an that promotes → in a mutagenesis study of the tetrahydrofuran moiety in simian kidney (COS) cells (21Takeshita M. Eisenberg W. Nucleic Acids Res. 1994; 22: 1897-1902Google Scholar), but not in a similar study of natural abasic sites Neto J.B. Cabral R.E.C. Margot A. Le Page F. Sarasin A. Gentil A. J. Mol. Biol. 1994; 240: 416-420Google Scholar). is that the structural and in simian kidney cells, but not in E. this is not by the in vitro reported The be by of the miscoding potential of the in mammalian cells. We conclude from these that the miscoding properties of natural abasic sites are similar, not to of the tetrahydrofuran analog. of the ring-opened form of the natural abasic site to block translesional synthesis and to predominantly cyclic and synthesis past the lesion. The order of nucleotide insertion > dGMP > > is similar for both DNA polymerases dAMP, positioned opposite or at the 3′-primer is extended than These kinetic are with that dAMP is preferentially incorporated into DNA by the Klenow fragment of pol I and by pol α. end addition of dAMP, observed in primer extension catalyzed by the exo− Klenow the that the A an of this and DNA